Ceramides and their use in diagnosing CVD

ABSTRACT

The present invention inter alia provides a method, and use thereof, of predicting CV complications such as AMI, ACS, stroke, and CV death by determining the concentrations of at least one ceramide of Group A and at least one ceramide of Group B in a biological sample and comparing those concentrations to a control. Finding a decreased concentration of at least one Group A ceramide and an increased concentration of at least one Group B ceramide indicates that the subject has an increased risk of developing one or more CV complications. Also provided are a newly identified subset of ceramide molecules, labelled versions thereof, and kits and compositions comprising the same for use in predicting and/or diagnosing CV complications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/159,650 filed 19 May 2016 (Allowed), which is a continuation of U.S.application Ser. No. 14/677,595 filed 2 Apr. 2015 (U.S. Pat. No.9,347,960), which claims priority to and the benefit of U.S. ProvisionalApplication No. 62/012,543 filed on 16 Jun. 2014, the entire contents ofwhich are incorporated herein by reference.

FIELD

This application relates generally to ceramide biomarkers and their usein identifying subjects having an increased risk for developingcardiovascular (CV) complications, such as AMI (acute myocardialinfarction), ACS (acute coronary syndrome), stroke or CV death.

BACKGROUND

Current CV markers, LDL-C, HDL-C, and total cholesterol, are not able toidentify coronary artery disease (CAD) patients or subjects that have anelevated risk for CV complications, such as AMI, ACS, stroke and CVdeath, from patients having more stable disease.

The term myocardial infarction pathologically denotes the death ofcardiac myocytes due to extended ischemia, which may be caused by anincrease in perfusion demand or a decrease in blood flow. The event iscalled “acute” if it is sudden and serious. Diagnosis of AMI isdetermined by a high clinical suspicion from history and physicalexamination, in addition to changes in cardiac biomarkers (creatininekinase MB [CK-MB], troponins, and myoglobin) and electrocardiogram (ECG)findings. Imaging techniques, such as two-dimensional echocardiography,are also useful in demonstrating myocardial dysfunction. Current CVmarkers, LDL-C, HDL-C, and total cholesterol, however, only identifywhen the damage of heart tissue has already occurred. They fail topredict the likelihood of the CVD complication occurring.

Acute coronary syndrome (ACS) is a term used for any condition broughton by sudden, reduced blood flow to the heart. The first sign of acutecoronary syndrome can be sudden stopping of the heart called cardiacarrest. Acute coronary syndrome is often diagnosed in an emergency roomor hospital with same cardiac biomarkers or electrocardiogram (ECG) asAMI, that provide evidence on damaged heart tissue or problems inheart's electric activity.

A stroke is the loss of brain function due to a disturbance in the bloodsupply to the brain, depriving brain tissue of oxygen and food. Withinminutes, brain cells begin to die. A stroke may be caused by a blockedartery (ischemic stroke) or a leaking or burst blood vessel (hemorrhagicstroke). Some people may experience a temporary disruption of blood flowthrough their brain (transient ischemic attack, or TIA). Strokes areusually diagnosed by brain imaging and carrying out physical tests.

Sudden cardiac death (SCD) is a sudden, unexpected death caused by lossof heart function, also named as sudden cardiac arrest (SCA). Suddencardiac arrest is not a heart attack (myocardial infarction). Heartattacks occur when there is a blockage in one or more of the coronaryarteries, preventing the heart from receiving enough oxygen-rich blood.In contrast, sudden cardiac arrest occurs when the electrical system tothe heart malfunctions and suddenly becomes very irregular. The heartbeats dangerously fast. Ventricular fibrillation may occur, and blood isnot delivered to the body. In the first few minutes, the greatestconcern is that blood flow to the brain will be reduced so drasticallythat a person will lose consciousness. Death follows unless emergencytreatment is begun immediately. Sudden cardiac arrest happens withoutwarning and is rarely diagnosed with medical tests as its happening.Instead, SCA often is diagnosed after it happens, by ruling out othercauses of a person's sudden collapse.

AMI, ACS, stroke and sudden cardiac death are diagnosed in acute stage,but predictive markers are not available. The risk factors behind theseevents are for example, age, hypolipidemia, hypertension, smoking,diabetes, CAD or previous heart attack. Yet, no diagnostic test thatcould predict the events exists, and cardiovascular diseases are theleading cause of death worldwide. Furthermore, CVD costs for societymore than any other group of diseases. The same tests that are used fordiagnosing CVD are utilized in predicting the events. Today the mostinnovative approach is to use LDL-C, HDL-C, Lp(a), Lp-PLA2 (PLAC test)or CRP. However, none of the listed lipid based markers (LDL-C, HDL-C,Lp(a), Lp-PLA2 (PLAC test)) provide clinically useful predictiveinformation allowing stratification aid to physicians. CRP has beenpromising in the research setting, however it has proven to beunspecific (CRP is an acute phase reactant that can react to manydifferent stimuli leading to highly variable test results) and thus CRPvalues are difficult to interpret in the clinical use. There is an unmetneed for a diagnostic test that could predict CV complications, such asAMI (acute myocardial infarction), ACS (acute coronary syndrome), strokeand CV death.

The ceramide based risk stratification offers superior p-values comparedto any other lipid based biomarker today. Furthermore, the levels ofplasma ceramides can be affected with specific lipid lowering treatments(such as statins) and, therefore, ceramide markers offer precise andactionable risk stratification.

A large group of lipid molecules, including certain ceramides, andratios calculated from two lipid molecules have been identified forpredicting CV outcomes in CAD patients who are undergoing statintreatment or who are not undergoing statin treatment (Zora Biosciencespatent applications WO2013068373 and WO2013068374) or for identifyinghigh-risk CAD patients or predicting whether a subject is at risk fordeveloping CV complications (Zora Biosciences patent application U.S.Ser. No. 13/695,766 and U.S. Ser. No. 13/805,319).

However, there remains a need for improved methods of predicting therisk of a patient developing a CV complication, such as AMI, ACS,stroke, and CV death.

SUMMARY

This application discloses a unique rule for selecting combinations ofceramides based on their structure and using them to predictCVD/CAD-associated complications, including AMI, ACS, stroke, and CVdeath. These ceramide markers thus provide a means to identify and treathigh-risk coronary artery disease patients. These sensitive and specificceramide markers were specifically tested to display superior diagnosticand prognostic value compared to the current clinically-used markerspredictive for CVD/CAD outcomes. Using combinations of ceramidebiomarkers according to the newly established rule will facilitateearlier intervention, less symptom development and suffering anddecreased morbidity/mortality associated with CVD. Thus, the combinationof ceramide markers described and claimed herein allow for individualtailoring of treatment, drug intervention and follow-up for patientsbeing at risk to develop major CV complications, such as AMI, ACS,stroke, and CV death.

According to this newly identified rule, certain ceramides have beenclassified into separate groups based on their structure and theirassociation, when combined, (based on either an increased or decreasedconcentration) with an increased risk to develop CV complications, suchas AMI, ACS, stroke, and CV death. Thus, in certain embodiments, themethods involve determining the concentration of at least one ceramideof Formula (I) (Group A ceramide) and at least one ceramide of Formula(II) (Group B ceramide) in a biological sample obtained from a subject.

A decreased concentration of at least one ceramide of Formula (I), whencombined with an increased concentration of at least one ceramide ofFormula (II) associates with an increased risk of a subject developing aCVD complication, such as AMI, ACS, stroke, and CV death.

Ceramides of Formula (I) have the following structure:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 21-25carbon atoms.

Ceramides of Formula (II) have the following structure:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 15 or 17carbon atoms, a mono-unsaturated alkyl chain having 21-23 carbon atoms,or a di-unsaturated alkyl chain having 23 or 25 carbon atoms.

In one embodiment, the method of determining whether a subject is atrisk to develop one or more CV complications comprises the steps of: a)determining the concentration of at least one Group A ceramide in abiological sample obtained from the subject, b) determining theconcentration of at least one Group B ceramide in the biological sampleobtained from the subject, c) comparing the concentration of the atleast one ceramide of Group A and the concentration of the at least oneceramide of Group B to a control sample; and d) determining that thesubject has an increased risk of developing one or more CVcomplications, if the sample contains a decreased concentration of theat least one ceramide of Group A and an increased concentration of theat least one ceramide of Group B, as compared to the control sample. Incertain embodiments, the method further comprises a step of spiking thebiological sample with at least one deuterium-labelled ceramide ofFormula I and at least one deuterium-labelled ceramide of Formula IIprior to determining the concentration of the at least one ceramide ofFormula I and II.

In one embodiment, the concentrations of the following ceramides ofFormula (II) are determined: Cer(d18:1/16:0), Cer(d18:1/18:0), andCer(d18:1/24:1). In another embodiment, the concentration of thefollowing ceramide of Formula (I): Cer(d18:1/24:0) and theconcentrations of the following ceramides of Formula (II):Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/24:1) are determined.

Another aspect is directed to a subset of Group A ceramides and/or asubset of Formula B ceramides that were previously not known to exist inhuman blood samples and their use in methods of determining whether asubject is at risk to develop one or more CV complications, such as AMI,ACS, stroke, and CV death. Thus, in certain embodiments, the methodsinvolve determining the concentration of at least one ceramide ofFormula (III) (Group C ceramide) and at least one ceramide of Formula(IV) (Group D ceramide) in a biological sample obtained from a subject.

A decreased concentration of at least one ceramide of Formula (III),when combined with an increased concentration of at least one ceramideof Formula (IV) associates with an increased risk of a subjectdeveloping a CV complication, such as AMI, ACS, stroke, and CV death.

Ceramides of Formula (III) have the following structure:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or adi-unsaturated alkyl chain having 15 carbon atoms, and wherein R² is asaturated alkyl chain having 21-23 or 25 carbon atoms.

Ceramides of Formula (IV) have the following structure:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms andR² is a saturated alkyl chain having 15 or 17 carbon atoms, amono-unsaturated alkyl chain having 23 carbon atoms, or a di-unsaturatedalkyl chain having 23 carbon atoms; or wherein R¹ is a di-unsaturatedalkyl chain having 15 carbon atoms and R² is a saturated alkyl chainhaving 15 or 17 carbon atoms, a mono-unsaturated alkyl chain having21-23 carbon atoms, or a di-unsaturated alkyl chain having 23 carbonatoms.

In one embodiment, the method of determining whether a subject is atrisk to develop one or more CV complications comprises the steps of: a)determining the concentration of at least one Group C ceramide in abiological sample obtained from the subject, b) determining theconcentration of at least one Group D ceramide in the biological sampleobtained from the subject, c) comparing the concentration of the atleast one ceramide of Group C and the concentration of the at least oneceramide of Group D to a control sample; and d) determining that thesubject has an increased risk of developing one or more CVcomplications, if the sample contains a decreased concentration of theat least one ceramide of Group C and an increased concentration of theat least one ceramide of Group D, as compared to the control sample. Incertain embodiments, the method further comprises a step of spiking thebiological sample with at least one deuterium-labelled ceramide ofFormula III and at least one deuterium-labelled ceramide of Formula IVprior to determining the concentration of the at least one ceramide ofFormula III and IV.

In certain embodiments, the concentrations of the at least one ceramideof Formula (I) and the at least one ceramide of Formula (II) or the atleast one ceramide of Formula (III) and the at least one ceramide ofFormula (IV) are determined according to the following equations:Z=(ceramide of Formula I)^(a)/(ceramide of Formula II)^(b) orZ=(ceramide of Formula III)^(a)/(ceramide of Formula IV)^(b), wherein a,bϵR.

Another aspect relates to a method for evaluating the effectiveness of atreatment of CVD and/or one or more of its complications, such as AMI,ACS, stroke, and CV death, in a subject, the method comprising the stepsof a) determining the concentration of at least one Group A ceramide (orat least one Group C ceramide) in a biological sample obtained from thesubject, b) determining the concentration of at least one Group Bceramide (or at least one Group D ceramide) in the biological sampleobtained from the subject, c) comparing the concentration of the atleast one ceramide of Group A (or at least one Group C ceramide) and theconcentration of the at least one ceramide of Group B (or at least oneGroup D ceramide) to a control sample; and d) determining that thetreatment is effective if the sample contains an equal or increasedconcentration of the at least one ceramide of Group A (or at least oneGroup C ceramide) and an equal or decreased concentration of the atleast one ceramide of Group B (or at least one Group D ceramide), ascompared to the control sample.

In certain embodiments, the method for evaluating the effectiveness of atreatment of atherosclerosis or CVD and/or one or more of itscomplications in the subject may further comprise after the determiningstep, changing, supplementing, or keeping the same an alreadyadministered treatment in the subject based on the concentrations of theat least one ceramide of Group A and the at least one ceramide of GroupB obtained in the determining step.

Yet another aspect relates to a method of choosing an appropriatetreatment of CVD and/or one or more of its complications, such as AMI,ACS, stroke, and CV death, in a subject, the method comprising the stepsof a) determining the concentration of at least one Group A ceramide (orat least one Group C ceramide) in a biological sample obtained from thesubject, b) determining the concentration of at least one Group Bceramide (or at least one Group D ceramide) in the biological sampleobtained from the subject, c) comparing the concentration of the atleast one ceramide of Group A (or at least one Group C ceramide) and theconcentration of the at least one ceramide of Group B (or at least oneGroup D ceramide) to a control sample; and d) determining that thesubject is in need of treatment or a change in, or supplementation of,an already administered treatment if the sample contains a decreasedconcentration of the at least one ceramide of Group A (or at least oneGroup C ceramide) and an increased concentration of the at least oneceramide of Group B (or at least one Group D ceramide), as compared tothe control sample.

In one embodiment, the treatment, the effectiveness of which is to beevaluated or which is to be chosen as appropriate in accordance with themethods described and claimed herein, is a lipid modifying treatment(e.g., statin or other lipid lowering drug as described elsewhere inthis application). In another embodiment, the method further comprises astep of administering to the subject the treatment that the subject isdetermined to be in need of in step (d).

Yet another aspect relates to a method of treating or preventing CVDand/or one or more of its complications, such as AMI, ACS, stroke, andCV death, in a subject, the method comprising administering to thesubject a therapeutically effective dose of a drug, wherein the drug isa statin; another lipid lowering drug selected from an HMG-CoA reductaseinhibitor other than a statin, niacin (nicotinic acid), a cholesterolabsorption inhibitor, a cholesteryl ester transfer protein (CETP), abile acid sequestrant, a fibrate, a phytosterol, and a PCSK9 inhibitor;or a modulator of lipid/lipid concentration ratios selected from a smallmolecule, an antibody, an antisense RNA, a small interfering RNA(siRNA), and a natural or modified lipid, and wherein beforeadministering the drug the subject has been identified as suffering fromor having an increased risk of developing a CVD complication, such asAMI, ACS, stroke, and CV death based on a decreased concentration of atleast ceramide of Group A (or at least one ceramide of Group C) and anincreased concentration of at least one ceramide of Group B (or at leastone ceramide of Group D) as compared to a control sample.

The concentrations of ceramides in the biological samples can bedetermined using any currently available technique or later developedtechnology. In certain embodiments, the concentrations of the ceramidesare determined using mass spectrometry. In certain embodiments, the massspectrometry instrument is coupled to a direct sample infusion method orto a high performance separation method.

The biological sample from the subject and the control sample ispreferably a blood sample, more preferably a blood plasma sample, oralso preferably a blood serum sample. In certain embodiments, the bloodsample is a blood spot dried on a filter. It may also be a fraction ofblood, blood plasma or blood serum, e.g., a lipid fraction thereof.Thus, in certain embodiments, the methods comprise a further step ofextracting the lipids from the biological sample before determining theconcentrations of the Group A, B, C and/or D ceramides. Alternatively,both the sample from the subject and the control sample may be a tissuesample, e.g., artery tissue, such as carotid artery tissue, or arteryplaque material, such as carotid artery plaque material.

Yet another aspect is a composition or kit for predicting CVcomplications or for performing any of the methods disclosed herein. Incertain embodiments, the composition or kit comprises at least oneisotope (e.g., deuterium)-labelled ceramide of Formula I and/or at leastone ceramide of Formula (II). In one embodiment, the kit comprises thefollowing isotope (e.g., deuterium)-labelled ceramides of Formula (II):Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/24:1). In anotherembodiment, the kit comprises the following isotope (e.g.,deuterium)-labelled ceramide of Formula (I): Cer(d18:1/24:0) and thefollowing isotope (e.g., deuterium)-labelled ceramides of Formula (II):Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/24:1).

Another embodiment is directed to a composition or kit comprising atleast one isotope (e.g., deuterium)-labelled ceramide of Formula (III)and/or at least one isotope (e.g., deuterium)-labelled ceramide ofFormula (IV).

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate certain embodiments, and togetherwith the written description, serve to explain certain principles of thecompositions and methods disclosed herein.

FIG. 1 shows the mean relative changes/differences (the difference ofcase versus control group means divided by the mean concentration of thecontrol group) of ten Group A ceramide species with the lowest Student'st-test p-values.

FIG. 2 shows the mean relative changes/differences (the difference ofcase versus control group means divided by the mean concentration of thecontrol group) of ten Group B ceramide species with the lowest Student'st-test p-values.

FIG. 3 shows the mean relative changes/differences (the difference ofcase versus control group means divided by the mean concentration of thecontrol group) of six ceramide species that do not fall within either ofthe Group A or Group B ceramides.

FIG. 4 provides a schematic diagram of a system according to someembodiments of the invention. In particular, this figure illustratesvarious hardware, software, and other resources that may be used inimplementations of computer system 106 according to disclosed systemsand methods.

DETAILED DESCRIPTION

Reference will now be made in detail to various exemplary embodiments,examples of which are illustrated in the accompanying drawings anddiscussed in the detailed description that follows. It is to beunderstood that the following detailed description is provided to givethe reader a fuller understanding of certain embodiments, features, anddetails of aspects of the invention, and should not be interpreted aslimiting the scope of the invention.

1. Abbreviations

Unless indicated otherwise, the abbreviations used in this applicationhave the following meanings: LDL-C: low density lipoprotein cholesterol;HDL-C: high density lipoprotein cholesterol; CVD: cardiovasculardisease; CV: cardiovascular; CAD: coronary artery disease; IVUS:intravascular ultrasound; NIRS: near infrared spectroscopy; OCT: Opticalcoherence tomography; CABG: Coronary artery bypass surgery; MS: massspectrometry; OR: odds ratio; SD: standard deviation; AUC: area undercurve; Sens: sensitivity; Spec: specificity; MRC %: Mean relativechange; p-val: P-value; q-val: and Q-value; Pr(>Chi): Chi-square;ICD-10: International Statistical Classification of Diseases and RelatedHealth Problems 10th Revision.

2. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The terms “subject,” “host,” “patient,” and “individual” are usedinterchangeably herein to refer to any mammalian subject for whomdiagnosis or therapy is desired, particularly humans. The subject mayhave previously suffered from a cardiovascular disease event such asangina pectoris, myocardial infarction or stroke. The CVD may or may notbe a result of atherosclerosis. Or the subject may be a healthyindividual with no previous signs of CVD.

Coronary vascular disease/cardiovascular disease (CVD) has its generalmeaning in the art and is used to classify numerous conditions thataffect the heart, heart valves, blood, and vasculature of the body,including CAD. In the present disclosure the terms CVD and CAD may beused interchangeably. Cardiovascular diseases include endothelialdysfunction, coronary artery disease, angina pectoris, myocardialinfarction, atherosclerosis, congestive heart failure, hypertension,cerebrovascular disease, stroke, transient ischemic attacks, deep veinthrombosis, peripheral artery disease, cardiomyopathy, arrhythmias,aortic stenosis, and aneurysm. Such diseases frequently involveatherosclerosis. In a preferred embodiment of the invention, thecardiovascular disease is a cardiovascular disease associated withatherosclerosis.

As used herein, the term “computer-implemented method” means a methodwhich utilizes a machine or apparatus to achieve its objective.

As used herein, the term “processor” means a device which is capable ofinterpreting and executing instructions. Specifically, a processoremploys logic circuitry to receive input data and provide theappropriate output data. Processors can communicate with each other viaa network.

As used in this application, a “Group A ceramide,” “ceramide of GroupA,” or the like means a ceramide of Formula (I):

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 21-25carbon atoms.

As used in this application, a “Group B ceramide,” “ceramide of GroupB,” or the like means a ceramide of Formula (II):

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 15 or 17carbon atoms, a mono-unsaturated alkyl chain having 21-23 carbon atoms,or a di-unsaturated alkyl chain having 23 or 25 carbon atoms.

As used in this application, a “Group C ceramide,” “ceramide of GroupC,” or the like means a ceramide of Formula (III):

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or adi-unsaturated alkyl chain having 15 carbon atoms, and wherein R² is asaturated alkyl chain having 21-23 or 25 carbon atoms.

As used in this application, a “Group D ceramide,” “ceramide of GroupD,” or the like means a ceramide of Formula (IV):

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms andR² is a saturated alkyl chain having 15 or 17 carbon atoms, amono-unsaturated alkyl chain having 23 carbon atoms, or a di-unsaturatedalkyl chain having 23 carbon atoms; or wherein R¹ is a di-unsaturatedalkyl chain having 15 carbon atoms and R² is a saturated alkyl chainhaving 15 or 17 carbon atoms, a mono-unsaturated alkyl chain having21-23 carbon atoms, or a di-unsaturated alkyl chain having 23 carbonatoms.

As used herein, a “lipid lowering drug” according to the invention ispreferably an HMG-CoA reductase inhibitor, niacin (nicotinic acid), acholesterol absorption inhibitor, a cholesteryl ester transfer protein(CETP) inhibitor, a bile acid sequestrant, a fibrate, a phytosterol or aPCSK9 inhibitor.

As used herein, a “cholesterol absorption inhibitor” is preferablyezetimibe or SCH-48461; a cholesteryl ester transfer protein (CETP)inhibitor is preferably evacetrapib, anacetrapib or dalcetrapib; a bileacid sequestrant is preferably colesevelam, cholestyramine orcolestipol; a fibrate is preferably fenofibrate, gemfibrozil,clofibrate, or bezafibrate, and the PCSK9 inhibitor is selected from aPCSK9 specific antibody, an siRNA, and a peptidomimetic.

3. Ceramide Structure and Nomenclature

Ceramide molecules consists of sphingoid base (SB) and a fatty acid (FA)chain. The structure of one representative ceramide molecule is shownbelow.

This Cer(d18:1/18:0) lipid is a Group B ceramide, where R¹ is amono-unsaturated alkyl chain having 15 carbon atoms and R² is asaturated alkyl chain having 17 carbon atoms.

The nomenclature of ceramides is typically presented as a first pair ofnumbers corresponding to the SB and a second pair of numberscorresponding to the FA. For example, in the Cer(d18:1/18:0) moleculeabove, the first number pair (d18:1) refers to the SB and the secondnumber pair (18:0) refers to the FA. In SB and FA nomenclature, thefirst number of each pair refers to the number of carbon atoms in the SBor FA chain, while the second number refers to the number ofcarbon-carbon double bonds of the SB or FA chain. Thus, inCer(d18:1/18:0), the SB has 18 carbon atoms and one carbon-carbon doublebond, while the FA has 18 carbon atoms and no carbon-carbon doublebonds. FAs can be saturated or unsaturated depending on whether theyhave double bonds in their structure. For example, the FA 16:1 is anunsaturated FA with 16 carbon atoms and one carbon-carbon double bond,while the FA 18:0 is a saturated FA with 18 carbon atoms and nocarbon-carbon double bonds.

By way of further example, the structure of Cer(d18:2/23:0) is asfollows:

This lipid is a Group A ceramide, where R¹ is a di-unsaturated alkylchain having 15 carbon atoms and R² is a saturated alkyl chain having 22carbon atoms.

By way of further example, the structure of Cer(d16:1/22:1) is asfollows:

This lipid is a Group B ceramide, where R¹ is a mono-unsaturated alkylchain having 13 carbon atoms and R² is a mono-unsaturated alkyl chainhaving 21 carbon atoms.

In the de novo ceramide synthesis, the first double bond to the SB isformed in carbon 4, forming SBs d16:1 and d18:1. The second double bondof the SB is most commonly located in carbon 14, forming SB d18:2.

The double bond in the FA chain may be in various positions depending onthe length of the FA chain and the number of carbon-carbon double bonds.In the 22:1 FA, the most common double bond position is in carbon 13. Inthe 23:1 FA, the most common double bond position is in carbon 14. Inthe 24:1 FA, the most common double bond position is in position 15. Inthe 24:2 and 26:2 FAs, the most common double bond positions are incarbons 5 and 9.

4. Diagnostic Methods

Disclosed herein are methods for predicting CV complications includingAMI, ACS, stroke and cardiovascular death, by measuring certaincombinations of ceramides in a biological sample. Ceramides have beenimplicated in the pathogenesis of CVD based on animal experiments, whilethe data in humans is largely lacking. We have recently shown theassociation of certain lipids, including certain distinct ceramidemolecules, with CV mortality (U.S. Ser. No. 13/695,766, U.S. Ser. No.13/805,319, WO2013068373, WO2013068374 and Tarasov et al. 2014).

For diagnostic use, a marker should have as high sensitivity andspecificity as possible. Sensitivity measures the proportion of casesthat are correctly classified as a case by the marker, and specificitymeasures the proportion of controls that are correctly classified as acontrol by the marker. For diagnostic use it is important that highenough percentage of subjects that are going to have a CV event in thefuture will be identified for targeted treatments at early stages.However, for limiting the treatment costs, markers with high specificityare preferred over markers with low specificity.

For example, a test with sensitivity of 0.7 would imply that among apopulation of 100 000 patients, that are going to face a CV event, 70000 would be identified as having high risk for the event by the test.With a specificity of 0.2, among a population of 100 000 patients withstable CVD but with no forthcoming severe events, only 20 000 would becorrectly identified of being low risk, while 80 000 would be falselyidentified of being at high risk. This would mean that the targetedtreatment cost for 80 000 patients would be used in vain. In contrast,if an alternative test with a specificity value of 0.8 for the sameevent had been used, only 20 000 patients would be falsely identifiedhaving high risk for severe event. Compared to the low specificity test,this would save the treatment costs for 60 000 patients.

This application discloses a rule for selecting a combination ofceramides to identify patients having increased risk for developing CVcomplications with significantly improved sensitivity and specificity.This improved sensitivity and specificity is determined by the structureof the ceramides that fall within two distinct classes, such that adecreased concentration of at least one ceramide from the firststructural class and an increased concentration of at least one ceramidefrom the second structural class, indicate an increased risk fordeveloping CV complications. Specific combinations of ceramide speciesfrom both classes, selected according to the disclosed rule, aresuperior in predicting CV complications compared to traditional riskmarkers or individual ceramide molecules or a ratio thereof.

The improved diagnostic cannot be achieved by randomly selecting acombination of ceramide species. Instead, the ceramide combinationsshould be selected according to the rule disclosed herein by combiningat least one ceramide with a very long saturated fatty acid and at leastone ceramide with either long chain saturated fatty acid or very longchain unsaturated fatty acid. Specifically selected combinations ofceramides showed improved diagnostic sensitivity and specificity ascompared to individual ceramides or ratios derived from two lipidmolecules. The disease associated specificity of these specificcombinations of ceramides is determined by the structure of the ceramidespecies and the biological function of various ceramide synthases thatmake the members of the ceramide class.

Concentrations of ceramides with very long saturated fatty acid chains(from 22:0 to 26:0) are lower in persons having an increased risk for CVcomplications (Group A ceramide). On the other hand, concentrations ofceramides with long chain saturated fatty acids (16:0 and 18:0) and verylong chain unsaturated fatty acid (from 22:1 to 26:2) are higher inpersons having an increased risk of CV complications (Group Bceramides). The diagnosis of CV complications improves when at least oneGroup A ceramide and at least one Group B ceramide are measured, insteadof selecting, for example, two Group A or Group B ceramides.

The two distinct classes of ceramides that were found to provideimproved diagnostic power when combined are shown in the table below,where the ceramide species for Group A include one of the listedsphingoid bases (SB) and one of the listed fatty acids (FA), and wherethe ceramide species for Group B include one of the listed sphingoidbases (SB) and one of the listed fatty acids (FA).

Group A (decreasing concentration) Ceramides with very long saturated FASB FA d16:1 + 22:0 d18:0 23:0 d18:1 24:0 d18:2 25:0 26:0

Group B (increasing concentration) Ceramides with long chain saturatedFA and very long chain unsaturated FA SB FA d16:1 + 16:0 d18:0 18:0d18:1 22:1 d18:2 23:1 24:1 24:2 26:2

It is also possible to define the two structural classes of ceramides bychemical formulae. The Group A ceramides (decreasing concentrationassociated with an increased risk of CV complications) have a structureaccording to Formula I:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 21-25carbon atoms.

The Group B ceramides (increasing concentration associated with anincreased risk of CV complications) have a structure according toFormula II:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 15 or 17carbon atoms, a mono-unsaturated alkyl chain having 21-23 carbon atoms,or a di-unsaturated alkyl chain having 23 or 25 carbon atoms.

The Group A and Group B ceramides can also be classified by the functionof the ceramide synthases that are responsible for their synthesis. Sixdifferent ceramide synthases (CerS1-6) have been characterized inhumans. Based on known specificity of ceramide synthases (CerS) for FAsubstrates, Group B ceramides contain ceramides exclusively produced byCerS1, S4, S5 and S6, that are known to synthesize ceramides with longFAs (14-18 carbons). Fatty acids with very-long FA (≥22 carbon atoms),synthesized by CerS2 and S3, are found in both Groups A and B. However,the very-long ceramide molecules in Group A are saturated, while thevery-long ceramides in Group B are unsaturated. Thus, the products ofCerS2 and S3 are placed into Groups A or B depending on the saturationlevel.

By categorizing certain ceramides into distinct groups based onstructure and/or the specificity of the ceramide synthases for fattyacid substrates found within the ceramides, this application discloses arule for selecting a combination of ceramides with significantlyimproved sensitivity and specificity for identifying patients havingincreased risk for developing CV complications.

In a particular embodiment, a method is provided for determining whethera subject is at risk to develop one or more CV complications, such asAMI, ACS, stroke, and CV death, wherein the method comprises

(a) determining in a biological sample obtained from the subject theconcentration of at least one ceramide of Formula I:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 21-25carbon atoms;

(b) determining in the biological sample from the subject theconcentration of at least one ceramide of Formula II:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 15 or 17carbon atoms, a mono-unsaturated alkyl chain having 21-23 carbon atoms,or a di-unsaturated alkyl chain having 23 or 25 carbon atoms;

(c) comparing the concentration of the at least one ceramide of FormulaI and the concentration of the at least one ceramide of Formula II to acontrol sample; and

(d) determining that the subject has an increased risk of developing oneor more CV complications, if the biological sample contains a decreasedconcentration of the at least one ceramide of Formula I and an increasedconcentration of the at least one ceramide of Formula II, as compared tothe control sample.

Another aspect relates to a method for evaluating the effectiveness of atreatment of CVD and/or one or more of its complications, such as AMI,ACS, stroke, and CV death, in a subject, the method comprising the stepsof

(a) determining in a biological sample obtained from the subject theconcentration of at least one ceramide of Formula I:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 21-25carbon atoms;

(b) determining in the biological sample from the subject theconcentration of at least one ceramide of Formula II:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 15 or 17carbon atoms, a mono-unsaturated alkyl chain having 21-23 carbon atoms,or a di-unsaturated alkyl chain having 23 or 25 carbon atoms;

(c) comparing the concentration of the at least one ceramide of FormulaI and the concentration of the at least one ceramide of Formula II to acontrol sample; and

(d) determining that the treatment is effective if the sample containsan equal or increased concentration of the at least one ceramide ofFormula I and an equal or decreased concentration of the at least oneceramide of Formula II, as compared to the control sample.

Yet another aspect relates to a method of choosing an appropriatetreatment of CVD and/or one or more of its complications, such as AMI,ACS, stroke, and CV death, in a subject, the method comprising:

(a) determining in a biological sample obtained from the subject theconcentration of at least one ceramide of Formula I:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 21-25carbon atoms;

(b) determining in the biological sample from the subject theconcentration of at least one ceramide of Formula II:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 15 or 17carbon atoms, a mono-unsaturated alkyl chain having 21-23 carbon atoms,or a di-unsaturated alkyl chain having 23 or 25 carbon atoms;

(c) comparing the concentration of the at least one ceramide of FormulaI and the concentration of the at least one ceramide of Formula II to acontrol sample; and

(d) determining that the subject is in need of treatment or a change in,or supplementation of, an already administered treatment if the samplecontains a decreased concentration of the at least one ceramide ofFormula I and an increased concentration of the at least one ceramide ofFormula II, as compared to the control sample.

In one embodiment of the aforementioned methods, the concentrations ofat least 2, at least 3, at least 4, at least 5, or at least 6 ceramidesof Formula I are determined. In another embodiment, the concentrationsof at least 2, at least 3, at least 4, at least 5, or at least 6ceramides of Formula II are determined. In yet another embodiment, theconcentrations of at least 2, at least 3, at least 4, at least 5, or atleast 6 ceramides of Formula I are determined and the concentrations ofat least 2, at least 3, at least 4, at least 5, or at least 6 ceramidesof Formula II are determined.

In one embodiment of the aforementioned methods, the concentrations ofat least one of the following ceramides of Formula (II) are determined:Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/24:1). In certainembodiments, the following Formula (II) ceramides or combinationsthereof may be determined in the aforementioned methods:

Formula (II) Formula (II) Formula (II) Cer(d18:1/16:0) Cer(d18:1/18:0)Cer(d18:1/24:1) Cer(d18:1/16:0) Cer(d18:1/18:0) Cer(d18:1/16:0)Cer(d18:1/24:1) Cer(d18:1/18:0) Cer(d18:1/24:1) Cer(d18:1/16:0)Cer(d18:1/18:0) Cer(d18:1/24:1)

In another embodiment, the concentration of the following ceramide ofFormula (I): Cer(d18:1/24:0) and the concentrations of at least one ofthe following ceramides of Formula (II): Cer(d18:1/16:0),Cer(d18:1/18:0), and Cer(d18:1/24:1) are determined. In certainembodiments, the concentrations of the following combinations of Formula(I) and Formula (II) ceramides may be determined in the aforementionedmethods:

Formula (I) Formula (II) Formula (II) Formula (II) Cer(d18:1/24:0)Cer(d18:1/16:0) Cer(d18:1/24:0) Cer(d18:1/18:0) Cer(d18:1/24:0)Cer(d18:1/24:1) Cer(d18:1/24:0) Cer(d18:1/16:0) Cer(d18:1/18:0)Cer(d18:1/24:0) Cer(d18:1/16:0) Cer(d18:1/24:1) Cer(d18:1/24:0)Cer(d18:1/18:0) Cer(d18:1/24:1) Cer(d18:1/24:0) Cer(d18:1/16:0)Cer(d18:1/18:0) Cer(d18:1/24:1)

Another aspect is directed to a subset of Group A ceramides and a subsetof Group B ceramides that were previously not known to exist in humanblood samples and their use in methods of determining whether a subjectis at risk to develop one or more CV complications, such as AMI, ACS,stroke, and CV death. The newly identified ceramides are listed in Table1 below:

TABLE 1 Newly Identified d16:1 and d18:2 Ceramides d16:1 ceramides d18:2ceramides Cer(d16:1/16:0) Cer(d18:2/16:0) Cer(d16:1/18:0)Cer(d18:2/18:0) Cer(d16:1/22:0) Cer(d18:2/22:0) Cer(d16:1/23:0)Cer(d18:2/22:1) Cer(d16:1/24:0) Cer(d18:2/23:0) Cer(d16:1/24:1)Cer(d18:2/23:1) Cer(d16:1/24:2) Cer(d18:2/24:0) Cer(d16:1/26:0)Cer(d18:2/24:1) Cer(d18:2/24:2) Cer(d18:2/26:0)

The newly identified ceramides that represent a subset of the Group Aceramides have the structure of Formula III:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or adi-unsaturated alkyl chain having 15 carbon atoms, and wherein R² is asaturated alkyl chain having 21-23 or 25 carbon atoms.

The newly identified ceramides that represent a subset of the Group Bceramides have the structure of Formula IV:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms andR² is a saturated alkyl chain having 15 or 17 carbon atoms, amono-unsaturated alkyl chain having 23 carbon atoms, or a di-unsaturatedalkyl chain having 23 carbon atoms; or wherein R¹ is a di-unsaturatedalkyl chain having 15 carbon atoms and R² is a saturated alkyl chainhaving 15 or 17 carbon atoms, a mono-unsaturated alkyl chain having21-23 carbon atoms, or a di-unsaturated alkyl chain having 23 carbonatoms.

Accordingly, certain embodiments are directed to a method fordetermining whether a subject is at risk to develop one or more CVcomplications, such as AMI, ACS, stroke, and CV death, wherein themethod comprises

(a) determining in a biological sample obtained from the subject theconcentration of at least one ceramide of Formula III:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or adi-unsaturated alkyl chain having 15 carbon atoms, and wherein R² is asaturated alkyl chain having 21-23 or 25 carbon atoms;

(b) determining in the biological sample from the subject theconcentration of at least one ceramide of Formula IV:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms andR² is a saturated alkyl chain having 15 or 17 carbon atoms, amono-unsaturated alkyl chain having 23 carbon atoms, or a di-unsaturatedalkyl chain having 23 carbon atoms; or wherein R¹ is a di-unsaturatedalkyl chain having 15 carbon atoms and R² is a saturated alkyl chainhaving 15 or 17 carbon atoms, a mono-unsaturated alkyl chain having21-23 carbon atoms, or a di-unsaturated alkyl chain having 23 carbonatoms;

(c) comparing the concentration of the at least one ceramide of FormulaIII and the concentration of the at least one ceramide of Formula IV toa control sample; and

(d) determining that the subject has an increased risk of developing oneor more CV complications, if the biological sample contains a decreasedconcentration of the at least one ceramide of Formula III and anincreased concentration of the at least one ceramide of Formula IV, ascompared to the control sample.

Another aspect relates to a method for evaluating the effectiveness of atreatment of CVD and/or one or more of its complications, such as AMI,ACS, stroke, and CV death, in a subject, the method comprising:

(a) determining in a biological sample obtained from the subject theconcentration of at least one ceramide of Formula III:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or adi-unsaturated alkyl chain having 15 carbon atoms, and wherein R² is asaturated alkyl chain having 21-23 or 25 carbon atoms;

(b) determining in the biological sample from the subject theconcentration of at least one ceramide of Formula IV:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms andR² is a saturated alkyl chain having 15 or 17 carbon atoms, amono-unsaturated alkyl chain having 23 carbon atoms, or a di-unsaturatedalkyl chain having 23 carbon atoms; or wherein R¹ is a di-unsaturatedalkyl chain having 15 carbon atoms and R² is a saturated alkyl chainhaving 15 or 17 carbon atoms, a mono-unsaturated alkyl chain having21-23 carbon atoms, or a di-unsaturated alkyl chain having 23 carbonatoms;

(c) comparing the concentration of the at least one ceramide of FormulaIII and the concentration of the at least one ceramide of Formula IV toa control sample; and

(d) determining that the treatment is effective if the sample containsan equal or increased concentration of the at least one ceramide ofFormula III and an equal or decreased concentration of the at least oneceramide of Formula IV, as compared to the control sample.

Yet another aspect relates to a method of choosing an appropriatetreatment of CVD and/or one or more of its complications, such as AMI,ACS, stroke, and CV death, in a subject, the method comprising the stepsof

(a) determining in a biological sample obtained from the subject theconcentration of at least one ceramide of Formula III:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or adi-unsaturated alkyl chain having 15 carbon atoms, and wherein R² is asaturated alkyl chain having 21-23 or 25 carbon atoms;

(b) determining in the biological sample from the subject theconcentration of at least one ceramide of Formula IV:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms andR² is a saturated alkyl chain having 15 or 17 carbon atoms, amono-unsaturated alkyl chain having 23 carbon atoms, or a di-unsaturatedalkyl chain having 23 carbon atoms; or wherein R¹ is a di-unsaturatedalkyl chain having 15 carbon atoms and R² is a saturated alkyl chainhaving 15 or 17 carbon atoms, a mono-unsaturated alkyl chain having21-23 carbon atoms, or a di-unsaturated alkyl chain having 23 carbonatoms;

(c) comparing the concentration of the at least one ceramide of FormulaIII and the concentration of the at least one ceramide of Formula IV toa control sample; and

(d) determining that the subject is in need of treatment or a change in,or supplementation of, an already administered treatment if the samplecontains a decreased concentration of the at least one ceramide ofFormula III and an increased concentration of the at least one ceramideof Formula IV, as compared to the control sample.

In one embodiment of the aforementioned methods, the concentrations ofat least 2, at least 3, at least 4, at least 5, or at least 6 ceramidesof Formula III are determined. In another embodiment, the concentrationsof at least 2, at least 3, at least 4, at least 5, or at least 6ceramides of Formula IV are determined. In yet another embodiment, theconcentrations of at least 2, at least 3, at least 4, at least 5, or atleast 6 ceramides of Formula III are determined and the concentrationsof at least 2, at least 3, at least 4, at least 5, or at least 6ceramides of Formula IV are determined.

In certain embodiments of the aforementioned methods, the concentrationsof the at least one ceramide of Formula (I) and the at least oneceramide of Formula (II) or the at least one ceramide of Formula (III)and the at least one ceramide of Formula (IV) are determined accordingto the following equations: Z=(ceramide of Formula I)^(a)/(ceramide ofFormula II)^(b) or Z=(ceramide of Formula III)^(a)/(ceramide of FormulaIV)^(b), wherein a, bϵR and (ceramide of Formula I), (ceramide ofFormula II), (ceramide of Formula III), and (ceramide of Formula IV)refer to the concentration of the ceramide of Formula I, theconcentration of the ceramide of Formula II, the concentration of theceramide of Formula III, and the concentration of the ceramide ofFormula IV, respectively. According to this embodiment the subject hasan increased risk of developing one or more CV complications, if thebiological sample contains an increased Z value, as compared to thecontrol sample. In other embodiment the equation is used for determiningthat the treatment is effective if the sample contains an equal ordecreased Z value, as compared to the control sample. In anotherembodiment the Z value is calculated for determining that the subject isin need of treatment or in need of adjusting the level of the treatmentaccording to the appropriate guidelines on treating cardiovascularpatients and the prescribed regimens and dosing therein if the samplecontains an increased Z value, as compared to the control sample.

It may be useful and even advantageous for the methods and usesdescribed herein to further comprise a step of determining the serumlevel of total cholesterol, low-density lipoprotein cholesterol (LDL-C),high-density lipoprotein cholesterol (HDL-C), Apolipoprotein A-I,Apolipoprotein A-II, and/or Apolipoprotein B (ApoB) and/orApolipoprotein C-III (ApoC-III) in a sample from said subject.Furthermore, according to one embodiment of the disclosed methods oruses, the subject is preferably one that does not have elevated serumlevels of one or more of total cholesterol, low-density lipoproteincholesterol (LDL-C), Apolipoprotein C-III (ApoC-III) or Apolipoprotein B(ApoB), or a decreased serum level of HDL-cholesterol (HDL-C).

5. Detection Methods

Also disclosed herein are methods of detecting in a biological sampleobtained from a subject the concentration of at least one ceramide ofFormula I and at least one ceramide of Formula II, wherein the methodcomprises:

(a) determining in a biological sample obtained from the subject theconcentration of at least one ceramide of Formula I:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 21-25carbon atoms;

(b) determining in the biological sample from the subject theconcentration of at least one ceramide of Formula II:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 15 or 17carbon atoms, a mono-unsaturated alkyl chain having 21-23 carbon atoms,or a di-unsaturated alkyl chain having 23 or 25 carbon atoms; andoptionally

(c) comparing the concentration of the at least one ceramide of FormulaI and the concentration of the at least one ceramide of Formula II to acontrol sample.

In one embodiment of the aforementioned methods, the concentrations ofat least 2, at least 3, at least 4, at least 5, or at least 6 ceramidesof Formula I are determined. In another embodiment, the concentrationsof at least 2, at least 3, at least 4, at least 5, or at least 6ceramides of Formula II are determined. In yet another embodiment, theconcentrations of at least 2, at least 3, at least 4, at least 5, or atleast 6 ceramides of Formula I are determined and the concentrations ofat least 2, at least 3, at least 4, at least 5, or at least 6 ceramidesof Formula II are determined.

In one embodiment of the aforementioned methods, the concentrations ofat least one of the following ceramides of Formula (II) are determined:Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/24:1). In certainembodiments, the following Formula (II) ceramides or combinationsthereof may be determined in the aforementioned detection methods:

Formula (II) Formula (II) Formula (II) Cer(d18:1/16:0) Cer(d18:1/18:0)Cer(d18:1/24:1) Cer(d18:1/16:0) Cer(d18:1/18:0) Cer(d18:1/16:0)Cer(d18:1/24:1) Cer(d18:1/18:0) Cer(d18:1/24:1) Cer(d18:1/16:0)Cer(d18:1/18:0) Cer(d18:1/24:1)

In another embodiment, the concentration of the following ceramide ofFormula (I): Cer(d18:1/24:0) and the concentrations of at least one ofthe following ceramides of Formula (II): Cer(d18:1/16:0),Cer(d18:1/18:0), and Cer(d18:1/24:1) are determined. In certainembodiments, the concentrations of the following combinations of Formula(I) and Formula (II) ceramides may be determined in the aforementioneddetection methods:

Formula (I) Formula (II) Formula (II) Formula (II) Cer(d18:1/24:0)Cer(d18:1/16:0) Cer(d18:1/24:0) Cer(d18:1/18:0) Cer(d18:1/24:0)Cer(d18:1/24:1) Cer(d18:1/24:0) Cer(d18:1/16:0) Cer(d18:1/18:0)Cer(d18:1/24:0) Cer(d18:1/16:0) Cer(d18:1/24:1) Cer(d18:1/24:0)Cer(d18:1/18:0) Cer(d18:1/24:1) Cer(d18:1/24:0) Cer(d18:1/16:0)Cer(d18:1/18:0) Cer(d18:1/24:1)

In one embodiment of the aforementioned methods, the biological sampleand the control sample comprise at least one isotope (e.g., deuterium)labelled ceramide of Formula I and at least one isotope (e.g.,deuterium) labelled ceramide Formula II. In general, theisotope-labelled ceramide will be the same ceramide that is beingdetected in the biological sample. For example, if the concentrations ofthe following ceramides of Formula II: Cer(d18:1/16:0), Cer(d18:1/18:0),and Cer(d18:1/24:1) and the concentration of the following ceramide ofFormula I: Cer(d18:1/24:0) are determined, the biological sample and thecontrol sample will comprise an isotope-labelled Cer(d18:1/16:0), anisotope-labelled Cer(d18:1/18:0), an isotope-labelled Cer(d18:1/24:1),and an isotope-labelled Cer(d18:1/24:0).

In one embodiment of the aforementioned methods, the subject previouslysuffered from a cardiovascular disease event such as angina pectoris,myocardial infarction or stroke.

Also disclosed herein are methods of detecting in a biological sampleobtained from a subject the concentration of at least one ceramide ofFormula III and at least one ceramide of Formula IV, wherein the methodcomprises:

(a) determining in a biological sample obtained from the subject theconcentration of at least one ceramide of Formula III:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or adi-unsaturated alkyl chain having 15 carbon atoms, and wherein R² is asaturated alkyl chain having 21-23 or 25 carbon atoms;

(b) determining in the biological sample from the subject theconcentration of at least one ceramide of Formula IV:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms andR² is a saturated alkyl chain having 15 or 17 carbon atoms, amono-unsaturated alkyl chain having 23 carbon atoms, or a di-unsaturatedalkyl chain having 23 carbon atoms; or wherein R¹ is a di-unsaturatedalkyl chain having 15 carbon atoms and R² is a saturated alkyl chainhaving 15 or 17 carbon atoms, a mono-unsaturated alkyl chain having21-23 carbon atoms, or a di-unsaturated alkyl chain having 23 carbonatoms; and optionally

(c) comparing the concentration of the at least one ceramide of FormulaIII and the concentration of the at least one ceramide of Formula IV toa control sample.

In one embodiment of the aforementioned methods, the concentrations ofat least 2, at least 3, at least 4, at least 5, or at least 6 ceramidesof Formula III are determined. In another embodiment, the concentrationsof at least 2, at least 3, at least 4, at least 5, or at least 6ceramides of Formula IV are determined. In yet another embodiment, theconcentrations of at least 2, at least 3, at least 4, at least 5, or atleast 6 ceramides of Formula III are determined and the concentrationsof at least 2, at least 3, at least 4, at least 5, or at least 6ceramides of Formula IV are determined.

In one embodiment of the aforementioned methods, the biological sampleand the control sample comprise at least one isotope (e.g., deuterium)labelled ceramide of Formula III and at least one isotope (e.g.,deuterium) labelled ceramide Formula IV. In general, theisotope-labelled ceramide will be the same ceramide that is beingdetected in the biological sample.

In one embodiment of the aforementioned methods, the subject previouslysuffered from a cardiovascular disease event such as angina pectoris,myocardial infarction or stroke.

6. Measuring Ceramide Concentrations

In connection with all aspects and embodiments described and claimedherein, the determination of the ceramide concentration is typicallyperformed using an assay. Collecting information on the concentration ofa ceramide from the sample of a subject and, where appropriate, acorresponding control sample, can be performed with various chemical andhigh-resolution analytical techniques. Suitable analytical techniquesinclude, but are not limited to, mass spectrometry and nuclear resonancespectroscopy. Any high-resolution technique capable of resolvingindividual ceramides or ceramide classes and providing structuralinformation of the same can be used to collect the information on theceramide marker in question, e.g., ceramide profile from the biologicalsample. Collecting the information on the ceramide marker with massspectrometry (MS) is one of the preferred embodiments of the disclosedmethods. The MS instrument can be coupled to a direct sample infusionmethod, such as a robotic nanoflow ion source device, or to a highperformance separation method such as high performance liquidchromatography (HPLC) or ultra performance liquid chromatography (UPLC).

Other methods can be used or combined with MS and/or HPLC/UPLC to detectthe ceramides of interest, including, for example, nuclear magneticresonance spectroscopy, liquid chromatography, thin-layerchromatography, gas-chromatography, fluorescence spectroscopy or dualpolarisation interferometry, and/or an immunoassay such as an ELISA.According to an alternative or further embodiment a ceramide in a samplecan be detected and/or quantified using a binding moiety capable ofspecifically binding the ceramide. The binding moiety can include, forexample, a member of a ligand-receptor pair, i.e., a pair of moleculescapable of having a specific binding interaction. The binding moiety canalso include, for example, a member of a specific binding pair, such asantibody-antigen, enzyme-substrate, nucleic acid-based ligands, otherprotein ligands, or other specific binding pairs known in the art.

In a preferred embodiment, the ceramide concentrations are measuredusing mass spectrometry (MS), wherein the MS instrument may be coupledto direct infusion methods or high performance separation methods suchas HPLC or UPLC.

7. Sample Preparation and Isotope-Labelled Ceramides

In accordance with the methods described and claimed herein, both thebiological sample from the subject and the control sample is preferablya blood sample, more preferably a blood plasma sample or a blood serumsample. The sample may also be a dried blood spot. It may also be afraction of blood, blood plasma or blood serum, e.g., a lipid fractionextracted from the sample. The blood sample can be taken in connectionwith, for example, measuring the cholesterol levels in the patients. Thecollected blood sample can be prepared and serum or plasma can beseparated with techniques well known to a person skilled in the art.Vena blood samples can be collected from patients using a needle and aBD Vacutainer® Plastic Tubes or Vacutainer® Plus Plastic Tubes (BDVacutainer® SST′ Tubes contain spray-coated silia and a polymer gel forserum separation). Serum can be separated from the collected bloodsample, for example, by centrifugation at 1300 RCF for 10 min at roomtemperature and stored in small plastic tubes at −80° C. Plasma can beseparated, for example, by centrifugation at 2,500×g at 4° C. for 5minutes. A dried blood spot is prepared by placing a spot of blood onfilter paper and allowing it to air dry.

Alternatively, both the sample from the subject and the control samplemay also be a tissue sample, e.g., artery tissue, such as carotid arterytissue, or artery plaque material, such as carotid artery plaquematerial.

In one embodiment, the biological sample is spiked with a known amountof a isotope-labelled Group A, Group B, Group C, or Group D ceramide.Any isotope that can be used to detect the ceramides of interest can beused, including but not limited to hydrogen (e.g., deuterium), carbon,and oxygen isotopes. In a preferred embodiment, the biological sample isspiked with the isotope-labelled ceramide prior to lipid extraction. Theisotope-labelled ceramide serves as an internal standard and is not anaturally occurring molecule. In a preferred embodiment, a deuteriumlabel is used to produce deuterium-labelled ceramides, including, butnot limited to N-palmitoyl-D-erythro-sphingosine-d7 (d18:1/16:0),N-stearoyl-D-erythro-sphingosine-d7 (d18:1/18:0),N-lignoceroyl-D-erythro-sphingosine-d7 (d18:1/24:0), andN-nervonoyl-D-erythro-sphingosine-d7 (Cer(d18:1/24:1)).

8. Controls

The ceramide levels in a biological sample obtained from a subject arecompared to a control. The control may be a biological sample from ahealthy individual. The control may also be a sample from CAD patient(s)with no history of major CVD events. It may also be a sample thatrepresents a combination of samples from a generalized population ofhealthy individuals or a sample that represents a combination of samplesfrom a CAD patient population with no history of major CVD events. Thebiological sample may be whole blood, blood serum, or blood plasma. Itmay also be a tissue sample. However, in a preferred embodiment, thebiological sample is plasma or serum.

Alternatively, the control may be a set of data concerning a ceramidemarker in accordance with the present invention, e.g., information onthe concentration of ceramide(s) in accordance with the presentinvention in a sample when taken from a healthy individual, or in acombination of samples when taken from a generalized population ofhealthy individuals, or from CAD patient(s) with no history of major CVDevents, or from a CAD patient population with no history of major CVDevents. The information, and thus the corresponding set of data, mayhave been previously determined, calculated or extrapolated, or may haveyet to be determined, calculated or extrapolated, or may also be takenfrom the literature.

In the methods of evaluating the effectiveness of a treatment, thecontrol sample may be from a healthy individual, as discussed above, inwhich case, finding that the biological sample from the subject containsconcentrations of at least one Group A (or Group C) ceramide and atleast one Group B (or Group D) ceramide that are about the same as orequal to the control sample (not significantly different) indicates thatthe treatment was effective. Alternatively, the control sample can beobtained from a subject who is at risk to develop one or more CVcomplications, such as AMI, ACS, stroke, or CV death. In certainembodiments, the subject from whom the control sample is obtained is thesame individual being treated, in which case a concentration of at leastone Group A (or Group C) ceramide that is increased relative to thecontrol sample and a concentration of at least one Group B (or Group D)ceramide that is decreased relative to the control sample, indicatesthat the treatment was effective.

9. Composition and Kits

Another aspect is a kit for predicting CV complications or forperforming any of the methods disclosed herein, wherein the kitcomprises at least one isotope (e.g., deuterium) labelled ceramide ofGroup A, B, C, or D, and optionally one or more reagents for performingthe method. Also encompassed are compositions comprising at least oneisotope (e.g., deuterium) labelled ceramide of Group A, B, C, or D.

In one embodiment, the composition or kit comprises at least one isotope(e.g., deuterium)-labelled ceramide of Formula I:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 21-25carbon atoms.

In another embodiment, the composition or kit comprises at least oneisotope (e.g., deuterium)-labelled ceramide of Formula II:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 15 or 17carbon atoms, a mono-unsaturated alkyl chain having 21-23 carbon atoms,or a di-unsaturated alkyl chain having 23 or 25 carbon atoms.

For example, in certain embodiments, the composition or kit comprisesone of the following isotope (e.g., deuterium)-labelled ceramides ofFormula II or combinations thereof:

Formula (II) Formula (II) Formula (II) Cer(d18:1/16:0) Cer(d18:1/18:0)Cer(d18:1/24:1) Cer(d18:1/16:0) Cer(d18:1/18:0) Cer(d18:1/16:0)Cer(d18:1/24:1) Cer(d18:1/18:0) Cer(d18:1/24:1) Cer(d18:1/16:0)Cer(d18:1/18:0) Cer(d18:1/24:1)

In another embodiment, the kit or composition comprises at least oneisotope (e.g., deuterium)-labelled ceramide of Formula I and at leastone isotope (e.g., deuterium)-labelled ceramide of Formula II. In otherembodiments, the kit or composition comprises at least 2, at least 3, atleast 4, at least 5, or at least 6 isotope (e.g., deuterium)-labelledceramides of Formula I and/or at least 2, at least 3, at least 4, atleast 5, or at least 6 isotope (e.g., deuterium)-labelled ceramides ofFormula II.

For example, in certain embodiments, the composition or kit comprisesthe following combinations of isotope (e.g., deuterium)-labelledceramides of Formula (I) and Formula II:

Formula (I) Formula (II) Formula (II) Formula (II) Cer(d18:1/24:0)Cer(d18:1/16:0) Cer(d18:1/24:0) Cer(d18:1/18:0) Cer(d18:1/24:0)Cer(d18:1/24:1) Cer(d18:1/24:0) Cer(d18:1/16:0) Cer(d18:1/18:0)Cer(d18:1/24:0) Cer(d18:1/16:0) Cer(d18:1/24:1) Cer(d18:1/24:0)Cer(d18:1/18:0) Cer(d18:1/24:1) Cer(d18:1/24:0) Cer(d18:1/16:0)Cer(d18:1/18:0) Cer(d18:1/24:1)

In other embodiments, the composition or kit comprises at least oneisotope (e.g., deuterium)-labelled ceramide of Formula III:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or adi-unsaturated alkyl chain having 15 carbon atoms, and wherein R² is asaturated alkyl chain having 21-23 or 25 carbon atoms.

Another aspect is directed to a composition or kit comprising at leastone isotope (e.g., deuterium)-labelled ceramide of Formula IV:

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms andR² is a saturated alkyl chain having 15 or 17 carbon atoms, amono-unsaturated alkyl chain having 23 carbon atoms, or a di-unsaturatedalkyl chain having 23 carbon atoms; or wherein R¹ is a di-unsaturatedalkyl chain having 15 carbon atoms and R² is a saturated alkyl chainhaving 15 or 17 carbon atoms, a mono-unsaturated alkyl chain having21-23 carbon atoms, or a di-unsaturated alkyl chain having 23 carbonatoms.

In another embodiment, the kit or composition comprises at least oneisotope (e.g., deuterium)-labelled ceramide of Formula III and at leastone isotope (e.g., deuterium)-labelled ceramide of Formula IV. In otherembodiments, the kit or composition comprises at least 2, at least 3, atleast 4, at least 5, or at least 6 isotope (e.g., deuterium)-labelledceramides of Formula III and/or at least 2, at least 3, at least 4, atleast 5, or at least 6 isotope (e.g., deuterium)-labelled ceramides ofFormula IV. The composition may further comprise a solution, such asdistilled water, a buffered solution, or other appropriate solvent.

All kits disclosed herein may be accompanied by instructions to use themfor predicting a CV complication, such as AMI, ACS, stroke, and CVdeath.

10. Computer-Implemented Diagnostic Methods

In accordance with all aspects and embodiments disclosed herein, themethods provided may be computer-implemented.

In one embodiment, any of the computer-implemented methods of theinvention may further comprise the steps of (i) obtaining by at leastone processor information reflecting the concentration of at least oneGroup A ceramide and at least one Group B ceramide (or at least oneGroup C ceramide and at least one Group D ceramide) in the biologicalsample; and (ii) outputting in user readable format the concentration ofat least one Group A ceramide and at least one Group B ceramide (or atleast one Group C ceramide and at least one Group D ceramide) in thebiological sample.

In another embodiment, the computer-implemented methods may furthercomprise a step of determining by at least one processor a percentagedifference between a control and the concentration of at least one GroupA ceramide and at least one Group B ceramide (or at least one Group Cceramide and at least one Group D ceramide) in the biological sample;and a step of outputting in user readable format the percentagedifference obtained in the determining step (iii).

In yet another embodiment, the computer-implemented methods may furthercomprise a step of determining whether a subject is at risk to developone or more CV complications based on the percentage difference obtainedin the outputting step.

In one embodiment of the computer-implemented methods, the at least oneGroup A ceramide comprises Cer(d18:1/24:0) and the at least one Group Bceramide comprises Cer(d18:1/16:0), Cer(d18:1/18:0), andCer(d18:1/24:1).

In embodiments as shown in FIG. 4, computer system 106 may include oneor more processors 110 coupled to random access memory operating undercontrol of or in conjunction with an operating system. The processor(s)110 in embodiments may be included in one or more servers, clusters, orother computers or hardware resources, or may be implemented usingcloud-based resources. The operating system may be, for example, adistribution of the Linux™ operating system, the Unix™ operating system,or other open-source or proprietary operating system or platform.Processor(s) 110 may communicate with data store 112, such as a databasestored on a hard drive or drive array, to access or store programinstructions other data.

Processor(s) 110 may further communicate via a network interface 108,which in turn may communicate via the one or more networks 104, such asthe Internet or other public or private networks, such that a query orother request may be received from client 102, or other device orservice. Additionally, processor(s) 110 may utilize network interface108 to send information, instructions, workflows query partialworkflows, or other data to a user via the one or more networks 104.Network interface 104 may include or be communicatively coupled to oneor more servers. Client 102 may be, e.g., a personal computer coupled tothe internet.

Processor(s) 110 may, in general, be programmed or configured to executecontrol logic and control operations to implement methods disclosedherein. Processors 110 may be further communicatively coupled (i.e.,coupled by way of a communication channel) to co-processors 114.Co-processors 114 can be dedicated hardware and/or firmware componentsconfigured to execute the methods disclosed herein. Thus, the methodsdisclosed herein can be executed by processor 110 and/or co-processors114.

Other configurations of computer system 106, associated networkconnections, and other hardware, software, and service resources arepossible.

The following Examples further define and describe embodiments herein.

EXAMPLES Example 1

Study patients.

The Corogene study is a prospective cohort study where 5000 consecutiveFinnish patients were assigned to diagnostic coronary angiogram in theregion of Helsinki University Central Hospital. In this study, 436 CADpatients with >50% stenosis in at least one coronary artery wereanalyzed. In this nested case-control study, one control subject wasmatched for each case. The cases were CAD patients that died due to CVreasons during an average follow-up of 2.5 years. The matching controlswere CAD patients, who did not die during the follow-up for any cause.The matching criteria included: Age, gender, body mass index, statinuse, smoking and type 2 diabetes.

Analytical Methods.

For quantification, ceramides were extracted using a modified Folchlipid extraction performed on a Hamilton Microlab Star robot, asdescribed in Jung H R et al., High throughput quantitative molecularlipidomics. Biochim Biophys Acta. 2011 November; 1811(11):925-34, whichis hereby incorporated by reference in its entirety. Samples were spikedwith known amounts of non-endogeneous synthetic internal standards.After lipid extraction, samples were reconstituted inchloroform:methanol (1:2, v/v) and a synthetic external standard waspost-extract spiked to the extracts. The extracts were stored at −20° C.prior to MS analysis.

Ceramides were analyzed on a hybrid triple quadrupole/linear ion trapmass spectrometer (5500 QTRAP) equipped with an ultra high pressureliquid chromatography (UHPLC) system (Eksigent ultraLC 100 system) usingmultiple reaction monitoring (MRM)-based method in negative ion modebased on the description by Sullards M C et al., Structure-specific,quantitative methods for analysis of sphingolipids by liquidchromatography-tandem mass spectrometry: “inside-out” sphingolipidomics.Methods Enzymol. 2007; 432:83-115.

The LC-MRM analysis was supplemented to include the precise measurementof new groups of (d16:1 and d18:2) ceramides not previously known toexist in human plasma (see Table 1). Their existence was verified inhuman plasma by sequential MS/MS analyses and retention time. In MS/MS,the identification was confirmed by the selective sphingosine fragmentions produced by the expected molecular ions, e.g. m/z of 236.25corresponding to d16:1, m/z of 262.25 corresponding to d18:2. Further,this was complemented by the systematic retention time shift. Thecharacteristic shifts confirmed the identity of the novel ceramidespecies. Their existence and ability to perform as biomarkers for thepurpose described in this application was surprising and unexpected.

Masses and counts of detected peaks by mass spectrometry were convertedinto a list of corresponding lipid names and concentrations. Calibrationlines were generated to determine the dynamic quantification range foreach lipid class monitored, e.g., the quantification limits. Internalstandards were used for quantifying endogenous lipid species.Calibration lines were used to determine the quantification limits ofthe method.

Stringent cutoff was applied for separating background noise from actuallipid peaks. Each sample was controlled and only accepted whenfulfilling the acceptance criteria. Masses and counts of detected peakswere converted into a list of corresponding lipid names. Lipids werenormalized to their respective internal standard and sample volume toretrieve their concentrations.

Targeted Ceramide Quantification of Cer(d18:1/16:0), Cer(d18:1/18:0),Cer(d18:1/24:0), and Cer(d18:1/24:1) molecules.

Ceramides were extracted using a modified Folch lipid extractionperformed on a Hamilton Microlab Star robot, as described in Jung et al.For quantification of the specific combination of Cer(d18:1/16:0),Cer(d18:1/18:0), Cer(d18:1/24:0), and Cer(d18:1/24:1), the biologicalsamples were spiked with a known amount of the followingdeuterium-labelled internal standards prior to lipid extraction:N-palmitoyl-D-erythro-sphingosine-d7 (Cer(d18:1/16:0)),N-stearoyl-D-erythro-sphingosine-d7 (Cer(d18:1/18:0)),N-lignoceroyl-D-erythro-sphingosine-d7 (Cer(d18:1/24:0)), andN-nervonoyl-D-erythro-sphingosine-d7 (Cer(d18:1/24:1)). Calibration linestandards were made in solution as no suitable lipid free matrix wasavailable. Known amount of analyte and stable isotope-labelled internalstandards was added to chloroform:methanol (1:2, v/v) to make thecalibration line points.

LC-MS/MS was conducted on hybrid triple quadrupole/linear ion trap massspectrometer (QTRAP 5500) equipped with an ultra-high pressure liquidchromatography (UHPLC) system (Eksigent ultraLC 100 system) usingmultiple reaction monitoring (MRM) based method in positive ion mode.

Sample extract (5 μL) was loaded on Waters Acquity BEH C18, 2.1×50 mmid. 1.7 μm connected to Waters Acquity BEH C18, 1.7 μm VanGuardPre-Column at column temperature of 60° C. The mobile phase A consistedof 10 mM ammonium acetate in HPLC grade water with 0.1% formic acid andmobile phase B. The flow rate of 0.5 μL/min with the following gradient:0.0-0.5 min, 85% B; 0.5-1.5 min, 85 to 100 & B; 1.5-4 min, 100% B;4.10-5 min, 85% B. The source temperature was set to 300° C.; ion spraywas 5000V; declustering potential 30 V; entrance potential was 10; gas 1and gas 2 setting were 50 and 30. Dwell time for each scan was 25 msecand collision energy was set at 40 V. Total acquisition time was 4.5min.

Data was acquired with Analyse software (version 1.6) and processed withMultiQuant (version 3.0). Calibration line curves were constructed byplotting the peak area ratio of analyte/internal standards against theactual concentration using, for example, weighted of 1/x² forCer(d18:1/16:0) and Cer(d18:1/18:0) and 1/y² for Cer(d18:1/24:1) andCer(d18:1/24:1). Peak area ratio of the samples were read off thecalibration lines.

The 7 deuterium in the sphingosine backbone of the labelled standardsprovide a mass shift of 7 Da in their sphingosine fragment ions uponMS/MS of the parent ion. The mass difference of the parent ion includingsphingosine fragment ion in the MRM analysis of the endogenous andinternal standard will be therefore be 7 Da respectively, e.g. MRMtransitions of Cer d18:1/16:0 and D7-Cer 18:1/16:0 are 538.5/264.2 and545.5/271.2.

The identical analytical behavior, such as extraction recovery,ionization and retention time, of the internal standard andcorresponding endogenous species including the mass shift, facilitatesthe precise measurement and quantification. The peak area ratio of theendogeneous ceramide to its internal standard in samples is read off acalibration line to provide the precise quantity of the endogenousceramide in samples. The calibration line is obtained by analyzing adilution series of the endogeneous ceramides mixed with a fixed amountof the deuterated standards by LC-MRM as described.

The deuterium labelled internal standards were specifically manufacturedfor this assay. Thus, the absolute quantification of these specificdeuterium-labelled ceramides has not been performed previously.

Statistical Methods.

Odds ratio quantitatively describes the association between thepresence/absence of a described cardiovascular outcome event and thepresence/absence of no event for stable CAD individuals in thepopulation. The odds ratio per standard deviation (OR per SD) quantifiesthe odds of positive prediction in case group divided by the odds ofpositive prediction in the control group as a response to a one standarddeviation change in the variable. Quartile odds ratios (OR 2-4) quantifythe ratio of positive outcomes to negative outcomes in differentquartiles relative to these ratios in the first quartile. Area under thereceiver operating characteristic curve (AUC) describes the overallpredictive performance of the marker. Its values are between 0 and 1,with 0.5 corresponding to random, 50/50 (e.g., coin flipping),predictions of the outcome, and higher values indicating betterprediction accuracy in terms of sensitivity (sens) and specificity(spec). Mean relative change (MRC %) is calculated as the differencebetween the mean values of the ceramide concentrations in the case andcontrol groups divided by the mean value in the control group. P-valueis calculated using Student's t-test on log-transformed data. Also falsediscover rate q-value estimates are calculated. These q-values estimatethe percentage of false positive findings among all the markers whosep-value is smaller than the p-value of the corresponding marker.

A logistic regression model for explaining case versus control groupingusing the lipid concentrations (either single or two lipids in themodel, corresponding to single marker versus two marker diagnosticmodel) was fit to the Corogene study population. A likelihood ratio testwith deviance statistics was estimated. Deviance measurements arecommonly used to compare models. Deviance is a quality of fit statisticfor a model that represents a measure of the lack of fit between modeland data. In general, the larger the deviance, the poorer the fit of thedata to the model. The deviance is usually not interpreted directly, butrather compared to deviance(s) from other models fitted to the samedata. See e.g., Nelder, J. A.; Wedderburn, (1972). “Generalized LinearModels.” Journal of the Royal Statistical Society. Series A (General)135 (3): 370-384.

The likelihood ratio test can be applied for nested models, that is, fortwo models, one with a larger number of variables and the other with asubset of variables of the larger model. The deviance statistics followsapproximately Chi-square distribution from which the statisticalsignificance can be estimated. Here p<0.05 has been used as a thresholdfor significant difference. If p>0.05, the two models do notsignificantly differ from each other in terms of discriminating casesfrom controls. In contrast, when p<0.05, the model with larger number ofvariables outperforms the smaller model. Likelihood ratio test fornested models using deviance statistics is an established method forcomparing models with different number of variables.

Results.

In the example study, the ceramide concentrations in CAD patients withCV events during a follow-up were compared to control subjects withoutevents. FIGS. 1 and 2 show the mean relative changes/differences (thedifference of case versus control group means divided by the meanconcentration of the control group) of ten Group A and ten Group Bceramide species with the lowest Student's t-test p-values. Thesedemonstrate the power (e.g., better odds ratios (AUC), or sensitivityand specificity, lower p values) of individual ceramide species todiscriminate high-risk patients (with forthcoming CV events) fromcontrol patients, as most of the ceramide species in either Group A orGroup B discriminate cases and control groups with p<0.05.

Ceramides that do not fall within either Group A or Group B were alsoanalyzed and it was found, in general, that such ceramides do notdiscriminate cases from controls with p<0.05 by Student's t-test onlog-transformed concentration estimates, as shown in FIG. 3.

Deviance statistics were used to analyze various combinations ofceramide concentrations in patients from the Corogene study and, inparticular, CAD patients with CV events during a follow-up as comparedto control subjects without events. The results of the deviancestatistics are summarized in Model Comparisons 1-9.

Example 2

Model Comparison 1.

In Model Comparison 1, a single ceramide species (either Group A or B)was compared to a combination of two ceramides, one from Group A and theother from Group B. In Model Comparison 1, the fact that Pr(>Chi)<0.05for the combined two marker model against either of the single markermodels, indicates that the discriminatory power of the combined model issignificantly better than that of the single marker models, as shown inTable 4 below.

TABLE 4 Model Comparison 1 Cer(d18:1/16:0) (B) vs. Cer(d18:1/24:0) (A)vs. Cer(d18:1/16:0) (B) + Cer(d18:1/24:0) (A) + Cer(d18:1/24:0) (A)Cer(d18:1/16:0) (B) Deviance Pr(>Chi) Deviance Pr(>Chi) −45.2 1.80*10⁻¹¹−77.0 2.20*10⁻¹⁶

Example 3

Model Comparison 2.

There is no particular reason to restrict the discriminatory model to acombination of only two ceramides. However, for practical purposes andfor cost limiting reasons, it is preferable to use as few ceramides aspossible. Thus, one has to weigh the benefit of increasing sensitivityand specificity against the additional cost of analyzing more molecularlipid species. In Model Comparison 2, a model with two ceramide specieswas compared against a model with three ceramide species, where thethree ceramide markers included at least one Group A ceramide and atleast one Group B ceramide. In Model Comparison 2, Pr(>Chi)<0.05 for thecombined three marker model against either of the double marker modelsindicates that the discriminatory power of the three marker model issignificantly better than that of the double marker models, as shown inTable 5.

TABLE 5 Model Comparison 2 Cer(d18:0/18:0) (B) + Cer(d18:0/18:0) (B) +Cer(d18:1/23:1) (B) vs. Cer(d18:2/25:0) (A) vs. Cer(d18:0/18:0) (B) +Cer(d18:0/18:0) (B) + Cer(d18:1/23:1) (B) + Cer(d18:2/25:0) (A) +Cer(d18:2/25:0) (A) Cer(d18:1/23:1) (B) Deviance Pr(>Chi) DeviancePr(>Chi) −42.4 7.30*10⁻¹¹ −33.3 7.70*10⁻⁰⁹

Example 4

Model Comparison 3:

Increasing the number of ceramide species in the model does notnecessarily mean that a better model will result. As an example of this,Model Comparison 3 shows a three ceramide model that is not better thana two ceramide model. In this case, a combination of two ceramides fromGroup B were compared to three ceramides from Group B. In ModelComparison 3, Pr(>Chi)>0.05 for the combined three marker model ascompared to a two marker model, indicates that the discriminatory powerof the three marker model is not significantly better than that of thedouble marker model, as shown in Table 6.

TABLE 6 Model Comparison 3 Cer(d18:1/23:1) (B) + Cer(d18:1/18:0) (B) vs.Cer(d18:1/23:1) (B) + Cer(d18:0/18:0) (B) + Cer(d18:1/18:0) (B) DeviancePr(>Chi) −2.3 0.1321

Thus, Model Comparison 3 demonstrates that merely increasing the numberof ceramides does not result in an improved model performance. Rather,the combined ceramides should be selected according to the guidanceprovided in this application, with at least one ceramide of Group A andat least one ceramide of Group B.

Example 5

Model Comparison 4.

An interesting combination of ceramide species is shown in ModelComparison 4. This combination includes three Group B ceramides:Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/24:1); and one Group Aceramide: Cer(d18:1/24:0). This particular combination appearssystematically in all analyzed study populations. In addition, theceramide species in Model Comparison 4 are abundant in human serum orplasma samples when compared to the abundance of ceramide species ingeneral. The model which combines the four ceramide species outperformsmodels with its single components in all cases (all Pr(>Chi)<0.05), asshown in Table 7.

TABLE 7 Model Comparison 4 Cer(d18:1/16:0) (B) + Cer(d18:1/18:0) (B) +Cer(d18:1/24:0) (A) + Cer(d18:1/24:1) (B) Deviance Pr(>Chi) vs.Cer(d18:1/16:0) −66.3 2.69*10⁻¹⁴ vs. Cer(d18:1/18:0) −59.9 6.06*10⁻¹³vs. Cer(d18:1/24:0) −98.1 2.20*10⁻¹⁶ vs. Cer(d18:1/24:1) −86.42.20*10⁻¹⁶

Example 6

Model Comparisons 5-7.

There may be a general perception that the diagnostic power of acombination of biomarkers will increase as the number of biomarkersanalyzed increases. As shown in Model Comparison 3, however, thisperception does not necessarily play out with ceramide biomarkers in thecontext of predicting CV complications. Model Comparisons 5-7 furtherdemonstrate that simply increasing the number of ceramide species in themodel, without applying the rule of selecting at least one Group Aceramide and at least one Group B ceramide, does not improve the modelperformance. Thus, Model Comparisons 5-7 demonstrate the lack ofdiagnostic power for randomly selecting ceramides as compared toselecting ceramides based on the newly identified rule of selecting atleast one Group A ceramide and at least one Group B ceramide.

In Model Comparison 5, combinations of ceramides that do not fall withinGroup A or Group B were analyzed. In Table 8, 15 model comparisons areshown with their deviance statistics and Chi-square p-value estimates(with p<0.05 denoting significant difference between the models). Ineach model comparison, random combinations of ceramides that do notinclude at least one Group A ceramide and at least one Group B ceramidewere compared. In all 15 examples with varying model sizes, the p-valuefor the deviance statistics is greater than 0.05, which means that themodel with more non AB ceramides is not significantly better indiscriminating the case-control grouping as compared to the model withfewer ceramides.

TABLE 8 Model Comparison 5 Comparison of combinations outside groups Aand B Deviance P-value Cer(d18:1/20:0) + Cer(d18:1/21:0) +Cer(d18:1/26:2) + Cer(d18:0/20:0) vs −8.08*10⁻⁰⁵ 0.992826Cer(d18:1/20:0) + Cer(d18:1/26:2) + Cer(d18:0/20:0) Cer(d18:1/21:0) +Cer(d18:1/20:0) + Cer(d18:1/26:2) vs Cer(d18:1/20:0) + Cer(d18:1/26:2)−0.00879 0.925325 Cer(d18:1/20:0) + Cer(d18:1/21:0) vs Cer(d18:1/20:0)−0.05299 0.817934 Cer(d18:0/20:0) + Cer(d18:1/21:0) + Cer(d18:1/20:0) vsCer(d18:0/20:0) + Cer(d18:1/20:0) −0.11153 0.738412 Cer(d18:1/20:0) +Cer(d18:1/21:0) + Cer(d18:1/26:2) + Cer(d18:0/20:0) vs −0.14819 0.700267Cer(d18:1/20:0) + Cer(d18:1/21:0) + Cer(d18:1/26:2) Cer(d18:1/20:0) +Cer(d18:0/20:0) + Cer(d18:1/26:2) vs Cer(d18:1/20:0) + Cer(d18:1/26:2)−0.1569 0.692029 Cer(d18:1/20:0) + Cer(d18:0/20:0) vs Cer(d18:1/20:0)−0.2269 0.63383 Cer(d18:0/20:0) + Cer(d18:1/21:0) + Cer(d18:1/20:0) vsCer(d18:1/21:0) + Cer(d18:1/20:0) −0.28544 0.59316 Cer(d18:1/26:2) +Cer(d18:0/20:0) + Cer(d18:1/21:0) vs Cer(d18:1/26:2) + Cer(d18:0/20:0)−0.59528 0.440384 Cer(d18:1/26:2) + Cer(d18:1/21:0) vs Cer(d18:1/26:2)−0.80246 0.370359 Cer(d18:1/26:2) + Cer(d18:0/20:0) + Cer(d18:1/21:0) vsCer(d18:1/26:2) + Cer(d18:1/21:0) −0.99255 0.319119 Cer(d18:1/20:0) +Cer(d18:1/21:0) + Cer(d18:1/26:2) + Cer(d18:0/20:0) vs −0.99354 0.318878Cer(d18:1/21:0) + Cer(d18:1/26:2) + Cer(d18:0/20:0) Cer(d18:1/26:2) +Cer(d18:0/20:0) vs Cer(d18:1/26:2) −1.19973 0.273375 Cer(d18:1/20:0) +Cer(d18:0/20:0) + Cer(d18:1/26:2) vs Cer(d18:0/20:0) + Cer(d18:1/26:2)−1.58874 0.207506 Cer(d18:1/21:0) + Cer(d18:1/20:0) + Cer(d18:1/26:2) vsCer(d18:1/21:0) + Cer(d18:1/26:2) −1.8379 0.175197

In Model Comparison 6, random combinations of Group A ceramides (withoutat least one Group B ceramide) were compared. In Table 9, 15 modelcomparisons are shown with their deviance statistics and Chi-squarep-value estimates (with p<0.05 denoting significant difference betweenthe models). In all 15 examples with varying model sizes, the p-valuefor the deviance statistics is greater than 0.05 which means that themodel with more Group A ceramides is not significantly better indiscriminating the case-control grouping as compared to the model withfewer Group A ceramides.

TABLE 9 Model Comparison 6 Comparison of combinations from Group ADeviance P-value Cer(d16:1/23:0) + Cer(d18:2/26:0) vs Cer(d18:2/26:0)−1.22*10⁻⁰⁵ 0.997211 Cer(d18:1/25:0) + Cer(d18:2/22:0) +Cer(d18:1/24:0) + Cer(d16:1/26:0) + Cer(d18:2/23:0) −0.00016 0.990018 vsCer(d18:1/25:0) + Cer(d18:2/22:0) + Cer(d16:1/26:0) + Cer(d18:2/23:0)Cer(d18:1/24:0) + Cer(d18:1/26:0) + Cer(d18:0/24:0) + Cer(d18:0/25:0) +Cer(d18:2/22:0) −0.00027 0.986808 vs Cer(d18:1/24:0) + Cer(d18:1/26:0) +Cer(d18:0/24:0) + Cer(d18:0/25:0) Cer(d18:2/14:0) + Cer(d18:1/25:0) +Cer(d18:2/26:0) vs Cer(d18:1/25:0) + Cer(d18:2/26:0) −0.00052 0.981846Cer(d18:2/26:0) + Cer(d18:1/24:0) + Cer(d18:2/23:0) + Cer(d18:1/26:0) vs−0.00124 0.971963 Cer(d18:2/26:0) + Cer(d18:1/24:0) + Cer(d18:1/26:0)Cer(d18:0/24:0) + Cer(d18:2/24:0) + Cer(d16:1/26:0) + Cer(d16:1/24:0) vs−0.00241 0.960812 Cer(d18:0/24:0) + Cer(d18:2/24:0) + Cer(d16:1/26:0)Cer(d16:1/22:0) + Cer(d18:1/24:0) + Cer(d18:2/26:0) + Cer(d18:2/24:0) +Cer(d18:1/23:0) −0.00382 0.950741 vs Cer(d18:1/24:0) + Cer(d18:2/26:0) +Cer(d18:2/24:0) + Cer(d18:1/23:0) Cer(d18:2/26:0) + Cer(d16:1/26:0) +Cer(d18:2/24:0) + Cer(d16:1/24:0) vs −0.00462 0.945827 Cer(d18:2/26:0) +Cer(d18:2/24:0) + Cer(d16:1/24:0) Cer(d16:1/26:0) + Cer(d18:2/26:0) +Cer(d18:2/14:0) + Cer(d18:2/24:0) vs −0.0068 0.934272 Cer(d16:1/26:0) +Cer(d18:2/26:0) + Cer(d18:2/24:0) Cer(d16:1/23:0) + Cer(d18:2/25:0) +Cer(d18:1/25:0) + Cer(d18:2/24:0) vs −0.00743 0.931329 Cer(d16:1/23:0) +Cer(d18:2/25:0) + Cer(d18:2/24:0) Cer(d18:2/23:0) + Cer(d16:1/22:0) +Cer(d18:1/24:0) + Cer(d16:1/26:0) + Cer(d18:2/26:0) −0.01043 0.918649 vsCer(d18:2/23:0) + Cer(d16:1/22:0) + Cer(d16:1/26:0) + Cer(d18:2/26:0)Cer(d18:2/24:0) + Cer(d16:1/26:0) + Cer(d18:0/25:0) + Cer(d18:2/22:0) vs−0.01459 0.903867 Cer(d18:2/24:0) + Cer(d18:0/25:0) + Cer(d18:2/22:0)Cer(d18:2/26:0) + Cer(d18:2/14:0) + Cer(d18:1/24:0) + Cer(d18:1/26:0) +Cer(d16:1/22:0) −0.0147 0.903505 vs Cer(d18:2/26:0) + Cer(d18:1/24:0) +Cer(d18:1/26:0) + Cer(d16:1/22:0) Cer(d16:1/24:0) + Cer(d18:2/24:0) +Cer(d18:2/22:0) + Cer(d16:1/26:0) vs −0.01491 0.902817 Cer(d16:1/24:0) +Cer(d18:2/24:0) + Cer(d18:2/22:0) Cer(d18:1/26:0) + Cer(d16:1/23:0) +Cer(d18:2/14:0) + Cer(d18:2/23:0) vs −0.01718 0.895724 Cer(d18:1/26:0) +Cer(d18:2/14:0) + Cer(d18:2/23:0)

In Model Comparison 7, random combinations of Group B ceramides (withoutat least one Group A ceramide) were compared. In Table 10, 15 modelcomparisons are shown with their deviance statistics and Chi-squarep-value estimates (with p<0.05 denoting significant difference betweenthe models). In all 15 examples with varying model sizes, the p-valuefor the deviance statistics is greater than 0.05 which means that themodel with more Group B ceramides is not significantly better indiscriminating the case-control grouping as compared to the model withfewer Group B ceramides.

TABLE 10 Model Comparison 7 Comparison of combinations from Group BDeviance P-value Cer(d16:1/16:0) + Cer(d18:1/24:1) + Cer(d18:0/18:0) +Cer(d18:0/24:2) + Cer(d18:1/16:0) vs −7.10*10⁻⁰⁶ 0.997874Cer(d16:1/16:0) + Cer(d18:1/24:1) + Cer(d18:0/18:0) + Cer(d18:1/16:0)Cer(d18:1/23:1) + Cer(d18:0/24:2) vs Cer(d18:1/23:1) −7.20*10⁻⁰⁶0.997859 Cer(d18:2/22:1) + Cer(d16:1/16:0) + Cer(d18:0/18:0) +Cer(d18:1/16:0) + Cer(d18:0/24:2) vs −7.08*10⁻⁰⁵ 0.993284Cer(d18:2/22:1) + Cer(d16:1/16:0) + Cer(d18:0/18:0) + Cer(d18:1/16:0)Cer(d18:1/24:1) + Cer(d18:2/24:2) + Cer(d18:0/24:1) + Cer(d18:1/23:1) +Cer(d18:1/18:0) vs −0.00025 0.98747 Cer(d18:1/24:1) + Cer(d18:2/24:2) +Cer(d18:1/23:1) + Cer(d18:1/18:0) Cer(d18:1/23:1) + Cer(d18:1/16:0) +Cer(d18:1/24:2) + Cer(d18:1/18:0) + Cer(d18:0/24:1) vs −0.00034 0.985232Cer(d18:1/16:0) + Cer(d18:1/24:2) + Cer(d18:1/18:0) + Cer(d18:0/24:1)Cer(d16:1/16:0) + Cer(d18:2/18:0) + Cer(d18:0/16:0) + Cer(d18:0/24:2) vs−0.00047 0.982751 Cer(d18:2/18:0) + Cer(d18:0/16:0) + Cer(d18:0/24:2)Cer(d18:0/16:0) + Cer(d18:2/24:2) + Cer(d16:1/16:0) + Cer(d18:0/24:2) +Cer(d18:1/24:1) vs −0.00084 0.97694 Cer(d18:0/16:0) + Cer(d18:2/24:2) +Cer(d16:1/16:0) + Cer(d18:1/24:1) Cer(d18:1/22:1) + Cer(d18:1/23:1) +Cer(d16:1/16:0) + Cer(d18:2/24:2) vs −0.00098 0.975028 Cer(d18:1/22:1) +Cer(d18:1/23:1) + Cer(d18:2/24:2) Cer(d18:0/16:0) + Cer(d18:2/18:0) +Cer(d18:1/24:1) + Cer(d18:0/24:1) vs −0.00129 0.971348 Cer(d18:0/16:0) +Cer(d18:1/24:1) + Cer(d18:0/24:1) Cer(d16:1/16:0) + Cer(d18:1/22:1) +Cer(d18:1/23:1) vs Cer(d18:1/22:1) + Cer(d18:1/23:1) −0.00141 0.970081Cer(d18:2/16:0) + Cer(d18:0/24:2) + Cer(d18:2/24:2) + Cer(d18:0/18:0) +Cer(d18:2/22:1) vs −0.00188 0.965379 Cer(d18:2/16:0) + Cer(d18:0/24:2) +Cer(d18:2/24:2) + Cer(d18:0/18:0) Cer(d18:2/24:2) + Cer(d18:1/18:0) +Cer(d18:2/22:1) + Cer(d18:2/16:0) + Cer(d18:1/23:1) vs −0.00229 0.961812Cer(d18:1/18:0) + Cer(d18:2/22:1) + Cer(d18:2/16:0) + Cer(d18:1/23:1)Cer(d18:2/18:0) + Cer(d16:1/16:0) + Cer(d18:0/18:0) + Cer(d18:0/16:0) +Cer(d18:1/24:1) vs −0.00321 0.9548 Cer(d18:2/18:0) + Cer(d18:0/18:0) +Cer(d18:0/16:0) + Cer(d18:1/24:1) Cer(d16:1/16:0) + Cer(d18:2/18:0) +Cer(d18:0/16:0) + Cer(d18:0/24:2) vs −0.00378 0.950985 Cer(d16:1/16:0) +Cer(d18:2/18:0) + Cer(d18:0/16:0) Cer(d16:1/16:0) + Cer(d18:0/24:1) +Cer(d18:0/16:0) + Cer(d18:1/22:1) vs −0.00529 0.941997 Cer(d18:0/24:1) +Cer(d18:0/16:0) + Cer(d18:1/22:1)

Example 7

Model Comparisons 8-9.

Model Comparisons 8-9 supplement Model Comparisons 1, 2, and 4,comparing combinations of at least one Group A and at least one Group Bceramide to combinations of either all Group A ceramides (ModelComparison 8) or all Group B ceramides (Model Comparison 9). These modelcomparisons demonstrate the diagnostic power of selecting ceramidesbased on the newly identified rule of having at least one Group Aceramide and at least one Group B ceramide.

In Model Comparison 8, combinations of at least one Group A ceramide andat least one Group B ceramide were compared to combinations of onlyGroup A ceramides. In Table 11, 30 model comparisons are shown withtheir deviance statistics and Chi-square p-value estimates (with p<0.05denoting significant difference between the models). In all 30 exampleswith varying model sizes, the p-value for the deviance statistics isless than 0.05 which means that the model with at least one Group Aceramide and at least one Group B ceramide is significantly better indiscriminating the case-control grouping as compared to the model withonly Group A ceramides.

TABLE 11 Model Comparison 8 Comparison of combinations from group A + Bvs A Deviance P-value A_Cer(d18:2/24:0) + A_Cer(d18:2/14:0) +B_Cer(d18:1/24:1) + B_Cer(d18:1/24:2) vs −98.1871 4.77*10⁻²²A_Cer(d18:2/24:0) + A_Cer(d18:2/14:0) A_Cer(d18:2/14:0) +A_Cer(d18:1/25:0) + B_Cer(d18:1/23:1) + B_Cer(d18:1/16:0) vs −96.47591.12*10⁻²¹ A_Cer(d18:2/14:0) + A_Cer(d18:1/25:0) A_Cer(d18:2/14:0) +A_Cer(d18:1/24:0) + B_Cer(d18:1/16:0) + B_Cer(d18:0/24:1) vs −95.37341.95*10⁻²¹ A_Cer(d18:2/14:0) + A_Cer(d18:1/24:0) A_Cer(d18:2/24:0) +A_Cer(d18:0/24:0) + B_Cer(d18:1/24:2) vs −87.6104 7.97*10⁻²¹A_Cer(d18:2/24:0) + A_Cer(d18:0/24:0) A_Cer(d18:1/25:0) +B_Cer(d18:1/23:1) + B_Cer(d18:1/24:2) vs A_Cer(d18:1/25:0) −91.92871.09*10⁻²⁰ A_Cer(d16:1/26:0) + A_Cer(d18:1/26:0) + B_Cer(d18:1/24:2) vs−85.5673 2.24*10⁻²⁰ A_Cer(d16:1/26:0) + A_Cer(d18:1/26:0)A_Cer(d18:1/23:0) + A_Cer(d18:2/23:0) + B_Cer(d18:1/24:2) +B_Cer(d18:1/18:0) vs −87.2167 1.15*10⁻¹⁹ A_Cer(d18:1/23:0) +A_Cer(d18:2/23:0) A_Cer(d18:1/24:0) + B_Cer(d18:1/24:2) +B_Cer(d18:2/16:0) vs A_Cer(d18:1/24:0) −86.8036 1.42*10⁻¹⁹A_Cer(d18:1/23:0) + B_Cer(d18:1/24:2) vs A_Cer(d18:1/23:0) −80.38573.08*10⁻¹⁹ A_Cer(d18:2/25:0) + B_Cer(d18:1/24:2) + B_Cer(d16:1/16:0) vsA_Cer(d18:2/25:0) −85.2126 3.14*10⁻¹⁹ A_Cer(d18:2/14:0) +A_Cer(d18:2/24:0) + B_Cer(d18:0/18:0) + B_Cer(d18:1/16:0) vs −85.20393.15*10⁻¹⁹ A_Cer(d18:2/14:0) + A_Cer(d18:2/24:0) A_Cer(d16:1/22:0) +A_Cer(d18:1/25:0) + B_Cer(d18:1/18:0) + B_Cer(d16:1/16:0) vs −84.53424.40*10⁻¹⁹ A_Cer(d16:1/22:0) + A_Cer(d18:1/25:0) A_Cer(d18:2/22:0) +A_Cer(d18:1/24:0) + B_Cer(d18:2/16:0) + B_Cer(d18:1/16:0) vs −83.78956.39*10⁻¹⁹ A_Cer(d18:2/22:0) + A_Cer(d18:1/24:0) A_Cer(d16:1/23:0) +A_Cer(d18:2/23:0) + B_Cer(d18:1/24:2) vs −78.7667 6.99*10⁻¹⁹A_Cer(d16:1/23:0) + A_Cer(d18:2/23:0) A_Cer(d18:1/23:0) +B_Cer(d18:1/24:2) + B_Cer(d18:0/24:1) vs A_Cer(d18:1/23:0) −83.17598.68*10⁻¹⁹ A_Cer(d18:1/23:0) + B_Cer(d18:1/24:2) + B_Cer(d18:1/24:1) vsA_Cer(d18:1/23:0) −82.1792 1.43*10⁻¹⁸ A_Cer(d18:1/23:0) +A_Cer(d18:2/22:0) + B_Cer(d18:1/18:0) vs −76.7722 1.92*10⁻¹⁸A_Cer(d18:1/23:0) + A_Cer(d18:2/22:0) A_Cer(d18:1/25:0) +B_Cer(d18:1/16:0) + B_Cer(d18:0/16:0) vs A_Cer(d18:1/25:0) −80.16053.92*10⁻¹⁸ A_Cer(d18:1/25:0) + B_Cer(d18:1/23:1) + B_Cer(d18:0/16:0) vsA_Cer(d18:1/25:0) −77.8771 1.23*10⁻¹⁷ A_Cer(d18:2/14:0) +B_Cer(d18:1/24:2) vs A_Cer(d18:2/14:0) −72.8176 1.42*10⁻¹⁷A_Cer(d18:2/22:0) + A_Cer(d18:2/14:0) + B_Cer(d18:0/18:0) +B_Cer(d18:2/24:2) vs −77.0652 1.84*10⁻¹⁷ A_Cer(d18:2/22:0) +A_Cer(d18:2/14:0) A_Cer(d18:1/25:0) + A_Cer(d18:0/25:0) +B_Cer(d18:1/16:0) + B_Cer(d18:0/24:2) vs −77.0528 1.85*10⁻¹⁷A_Cer(d18:1/25:0) + A_Cer(d18:0/25:0) A_Cer(d18:2/24:0) +A_Cer(d18:2/14:0) + B_Cer(d18:1/24:1) + B_Cer(d18:1/23:1) vs −76.94151.96*10⁻¹⁷ A_Cer(d18:2/24:0) + A_Cer(d18:2/14:0) A_Cer(d18:2/22:0) +A_Cer(d16:1/23:0) + B_Cer(d18:1/24:2) + B_Cer(d18:0/24:1) vs −76.29662.71*10⁻¹⁷ A_Cer(d18:2/22:0) + A_Cer(d16:1/23:0) A_Cer(d18:1/24:0) +A_Cer(d18:2/22:0) + B_Cer(d18:1/24:1) vs −71.4015 2.91*10⁻¹⁷A_Cer(d18:1/24:0) + A_Cer(d18:2/22:0) A_Cer(d18:2/24:0) +A_Cer(d18:1/24:0) + B_Cer(d16:1/16:0) + B_Cer(d18:1/16:0) vs −75.98183.17*10⁻¹⁷ A_Cer(d18:2/24:0) + A_Cer(d18:1/24:0) A_Cer(d18:1/23:0) +A_Cer(d18:0/24:0) + B_Cer(d18:1/18:0) + B_Cer(d18:2/16:0) vs −75.44044.15*10⁻¹⁷ A_Cer(d18:1/23:0) + A_Cer(d18:0/24:0) A_Cer(d16:1/22:0) +A_Cer(d18:2/26:0) + B_Cer(d16:1/16:0) + B_Cer(d18:1/18:0) vs −75.31284.43*10⁻¹⁷ A_Cer(d16:1/22:0) + A_Cer(d18:2/26:0) A_Cer(d18:1/26:0) +B_Cer(d18:1/18:0) + B_Cer(d18:2/18:0) vs A_Cer(d18:1/26:0) −74.83765.61*10⁻¹⁷ A_Cer(d18:1/25:0) + A_Cer(d18:0/24:0) + B_Cer(d18:0/24:1) +B_Cer(d18:1/23:1) vs −73.7486 9.68*10⁻¹⁷ A_Cer(d18:1/25:0) +A_Cer(d18:0/24:0)

In Model Comparison 9, combinations of at least one Group A ceramide andat least one Group B ceramide were compared to combinations of onlyGroup B ceramides. In Table 12, 30 model comparisons are shown withtheir deviance statistics and Chi-square p-value estimates (with p<0.05denoting significant difference between the models). In all 30 exampleswith varying model sizes, the p-value for the deviance statistics isless than 0.05 which means that the model with at least one Group Aceramide and at least one Group B ceramide is significantly better indiscriminating the case-control grouping as compared to the model withonly Group B ceramides.

TABLE 12 Model Comparison 9 Comparison of combinations from groups A + Bvs B Deviance P-value A_Cer(d18:2/26:0) + A_Cer(d16:1/22:0) +B_Cer(d16:1/16:0) + B_Cer(d18:2/24:2) vs −87.9369 8.03*10⁻²⁰B_Cer(d16:1/16:0) + B_Cer(d18:2/24:2) A_Cer(d18:2/24:0) +A_Cer(d18:1/26:0) + B_Cer(d18:1/24:2) vs B_Cer(d18:1/24:2) −87.36351.07*10⁻¹⁹ A_Cer(d18:2/24:0) + A_Cer(d18:1/26:0) + B_Cer(d18:1/24:2) vsB_Cer(d18:1/24:2) −87.3635 1.07*10⁻¹⁹ A_Cer(d18:2/24:0) +A_Cer(d18:1/26:0) + B_Cer(d18:1/16:0) + B_Cer(d18:2/24:2) vs −84.63874.18*10⁻¹⁹ B_Cer(d18:1/16:0) + B_Cer(d18:2/24:2) A_Cer(d18:2/25:0) +B_Cer(d18:2/24:2) vs B_Cer(d18:2/24:2) −69.1183 9.27*10⁻¹⁷A_Cer(d16:1/22:0) + A_Cer(d18:2/25:0) + B_Cer(d18:2/16:0) +B_Cer(d18:2/24:2) vs −73.37 1.17*10⁻¹⁶ B_Cer(d18:2/16:0) +B_Cer(d18:2/24:2) A_Cer(d18:2/24:0) + B_Cer(d18:2/24:2) vsB_Cer(d18:2/24:2) −66.7609 3.07*10⁻¹⁶ A_Cer(d18:2/25:0) +A_Cer(d16:1/23:0) + B_Cer(d18:0/24:2) + B_Cer(d18:2/24:2) vs −70.74314.35*10⁻¹⁶ B_Cer(d18:0/24:2) + B_Cer(d18:2/24:2) A_Cer(d18:2/24:0) +B_Cer(d18:1/24:2) vs B_Cer(d18:1/24:2) −65.5054 5.80*10⁻¹⁶A_Cer(d18:1/23:0) + A_Cer(d18:2/26:0) + B_Cer(d18:1/16:0) +B_Cer(d18:1/24:2) vs −70.074 6.08*10⁻¹⁶ B_Cer(d18:1/16:0) +B_Cer(d18:1/24:2) A_Cer(d18:2/14:0) + A_Cer(d18:2/26:0) +B_Cer(d18:0/24:1) + B_Cer(d18:1/24:2) vs −68.4406 1.38*10⁻¹⁵B_Cer(d18:0/24:1) + B_Cer(d18:1/24:2) A_Cer(d16:1/24:0) +A_Cer(d18:1/23:0) + B_Cer(d16:1/16:0) vs B_Cer(d16:1/16:0) −68.07551.65*10⁻¹⁵ A_Cer(d18:2/24:0) + A_Cer(d18:2/25:0) + B_Cer(d18:1/23:1) +B_Cer(d18:1/24:1) vs −67.43 2.28*10⁻¹⁵ B_Cer(d18:1/23:1) +B_Cer(d18:1/24:1) A_Cer(d18:1/26:0) + B_Cer(d18:1/23:1) +B_Cer(d18:1/16:0) vs −61.4525 4.54*10⁻¹⁵ B_Cer(d18:1/23:1) +B_Cer(d18:1/16:0) A_Cer(d18:2/24:0) + A_Cer(d18:2/23:0) +B_Cer(d18:1/24:2) vs B_Cer(d18:1/24:2) −65.9445 4.79*10⁻¹⁵A_Cer(d18:2/25:0) + A_Cer(d18:0/24:0) + B_Cer(d18:1/24:1) vsB_Cer(d18:1/24:1) −65.7703 5.23*10⁻¹⁵ A_Cer(d18:0/24:0) +A_Cer(d18:1/25:0) + B_Cer(d18:1/24:2) + B_Cer(d18:2/22:1) vs −64.44921.01*10⁻¹⁴ B_Cer(d18:1/24:2) + B_Cer(d18:2/22:1) A_Cer(d18:1/26:0) +B_Cer(d18:1/24:2) vs B_Cer(d18:1/24:2) −59.7105 1.10*10⁻¹⁴A_Cer(d18:0/25:0) + A_Cer(d18:1/25:0) + B_Cer(d18:1/23:1) +B_Cer(d18:0/16:0) vs −63.3033 1.79*10⁻¹⁴ B_Cer(d18:1/23:1) +B_Cer(d18:0/16:0) A_Cer(d18:1/25:0) + A_Cer(d18:2/25:0) +B_Cer(d18:1/24:2) + B_Cer(d18:2/24:2) vs −62.2116 3.10*10⁻¹⁴B_Cer(d18:1/24:2) + B_Cer(d18:2/24:2) A_Cer(d18:2/14:0) +A_Cer(d18:2/25:0) + B_Cer(d18:1/16:0) + B_Cer(d18:2/18:0) vs −61.17785.19*10⁻¹⁴ B_Cer(d18:1/16:0) + B_Cer(d18:2/18:0) A_Cer(d18:1/26:0) +B_Cer(d18:1/23:1 ) + B_Cer(d18:1/24:1) vs −56.5949 5.35*10⁻¹⁴B_Cer(d18:1/23:1) + B_Cer(d18:1/24:1) A_Cer(d18:1/25:0) +B_Cer(d18:0/24:2) + B_Cer(d18:1/24:1) vs −55.574 9.00*10⁻¹⁴B_Cer(d18:0/24:2) + B_Cer(d18:1/24:1) A_Cer(d18:2/24:0) +B_Cer(d18:2/24:2) + B_Cer(d18:1/23:1) vs −55.4461 9.61*10⁻¹⁴B_Cer(d18:2/24:2) + B_Cer(d18:1/23:1) A_Cer(d18:2/24:0) +A_Cer(d18:0/24:0) + B_Cer(d18:2/18:0) vs B_Cer(d18:2/18:0) −59.93929.65*10⁻¹⁴ A_Cer(d18:0/24:0) + A_Cer(d18:1/25:0) + B_Cer(d18:0/16:0) vsB_Cer(d18:0/16:0) −59.55 1.17*10⁻¹³ A_Cer(d18:1/23:0) +A_Cer(d18:2/24:0) + B_Cer(d18:2/16:0) vs B_Cer(d18:2/16:0) −58.87061.65*10⁻¹³ A_Cer(d18:2/25:0) + B_Cer(d18:2/16:0) + B_Cer(d18:0/24:2) vs−54.189 1.82*10⁻¹³ B_Cer(d18:2/16:0) + B_Cer(d18:0/24:2)A_Cer(d18:2/26:0) + A_Cer(d18:2/22:0) + B_Cer(d18:1/24:1) vsB_Cer(d18:1/24:1) −58.6415 1.85*10⁻¹³ A_Cer(d18:2/24:0) +A_Cer(d18:0/25:0) + B_Cer(d16:1/16:0) + B_Cer(d18:0/18:0) vs −57.69352.96*10⁻¹³ B_Cer(d16:1/16:0) + B_Cer(d18:0/18:0)

Thus, the results of this study demonstrate at least the followingtrends: (1) combining at least one Group A ceramide and at least oneGroup B ceramide significantly improves their discriminatory power ascompared to a Group A ceramide or Group B ceramide alone; (2) increasingthe number of Group A and Group B ceramides significantly increases thediscriminatory power if the combination of ceramides includes at leastone Group A and at least one Group B ceramide; (3) increasing the numberof Group A ceramides (without a Group B ceramide) does not significantlyimprove the discriminatory power of the combined ceramides; (4)increasing the number of Group B ceramides (without a Group A ceramide)does not significantly improve the discriminatory power of the combinedceramides; and (5) increasing the number of ceramides that do not fallwithin either Group A or Group B does not significantly improve thediscriminatory power of the combined ceramides.

Accordingly, these data have helped to establish a new rule forselecting ceramide biomarkers to predict CV complications withsignificantly improved discriminatory power (discriminating patients atrisk to develop CV complications from control patients). By followingthe new selection rule and combining at least one Group A ceramide withat least one Group B ceramide, the case control grouping can besignificantly more accurate as compared to individual markers or lipidratios or combinations of ceramide markers that are not selectedaccording to this selection rule.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims

The invention claimed is:
 1. A method for evaluating the effectivenessof a treatment of CVD and/or one or more of its complications in a humansubject, wherein the method comprises: (a) determining in a biologicalsample obtained from the human subject the concentration of at least oneceramide of Formula (I):

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 21-25carbon atoms, and wherein the biological sample is a blood sample, bloodserum sample, a blood plasma sample, or a lipoprotein fraction obtainedtherefrom; (b) determining in the biological sample from the humansubject the concentration of at least one ceramide of Formula (II):

wherein R¹ is a mono-unsaturated alkyl chain having 13 carbon atoms or asaturated, mono-unsaturated or di-unsaturated alkyl chain having 15carbon atoms, and wherein R² is a saturated alkyl chain having 15 or 17carbon atoms, a mono-unsaturated alkyl chain having 21-23 carbon atoms,or a di-unsaturated alkyl chain having 23 or 25 carbon atoms; (c)comparing the concentration of the at least one ceramide of Formula (I)and the concentration of the at least one ceramide of Formula (II) to acontrol sample, and (d) determining that the treatment is effective ifthe sample contains an increased concentration of the at least oneceramide of Formula (I) and a decreased concentration of the at leastone ceramide of Formula (II), as compared to the control sample, whereinthe method further comprises spiking the biological sample with at leastone isotope-labelled ceramide of Formula (I) and at least oneisotope-labelled ceramide of Formula (II) prior to determining theconcentration of the at least one ceramide of Formula (I) and the atleast one ceramide of Formula (II) and wherein the one or more CVDcomplications comprise one or more of acute myocardial infarction (AMI),acute coronary syndrome (ACS), stroke and cardiovascular death.
 2. Themethod of claim 1, wherein the concentrations of the at least oneceramide of Formula (I) and the at least one ceramide of Formula (II)are determined using a mass spectrometry instrument, and optionallywherein the mass spectrometry instrument coupled to a direct sampleinfusion method or to a high-performance separation method.
 3. Themethod of claim 1, wherein concentrations of at least 2, at least 3, atleast 4, at least 5 or at least 6 ceramides of Formula (I) and/or atleast 2, at least 3, at least 4, at least 5 or at least 6 ceramides ofFormula (II) are determined.
 4. The method of claim 1, wherein theconcentrations of the following ceramides of Formula (II) aredetermined: Cer(d18:1/16:0), Cer(d18:1/18:0) and/or Cer(d18:1/24:1)and/or wherein the concentration of the following ceramide of Formula(I) is determined: Cer(d18:1/24:0).
 5. The method of claim 1, furthercomprising a step of extracting lipids from the biological sample priorto determining the concentration of the at least one ceramide of Formula(I) and the at least one ceramide of Formula (II).
 6. The method ofclaim 1, wherein the isotope is deuterium.
 7. The method of claim 1,further comprising determining the blood, serum or plasma level of totalcholesterol, low-density lipoprotein cholesterol (LDL-C), high-densitylipoprotein cholesterol (HDL-C), Apolipoprotein A-I (ApoAI),Apolipoprotein A-II (ApoAII), Apolipoprotein B (ApoB) and/orApolipoprotein C-III (ApoC-III) in a sample from said subject.
 8. Themethod of claim 1, further comprising, after the determining step,changing, supplementing, or keeping the same, an already administeredtreatment in the human subject from whom the biological sample wasobtained based on the concentrations of the at least one ceramide ofFormula (I) and the at least one ceramide of Formula (II).
 9. The methodof claim 1, wherein the human subject is undergoing a lipid-loweringdrug treatment and optionally wherein the lipid-lowering drug treatmentis a statin treatment.
 10. The method of claim 1, wherein the methodfurther comprises: (e) administering a treatment to the subject if thedetermining steps indicate the treatment is not effective.
 11. Themethod of claim 1, further comprising individual tailoring of treatment,life counseling, drug intervention and/or follow-up for the humansubject from whom the biological sample was obtained if the determiningsteps indicate the treatment is not effective.